Tissue specific reduction of lignin

ABSTRACT

The present invention provides an expression cassette comprising a polynucleotide that encodes a protein that diverts a monolignol precursor from a lignin biosynthesis pathway in the plant, which is operably linked to a heterologous promoter. Also provided are methods of engineering a plant having reduced lignin content, as well as plant cells, plant parts, and plant tissues from such engineered plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation application of U.S. application Ser.No. 16/537,416, filed Aug. 9, 2019, which is a divisional application ofU.S. application Ser. No. 14/774,614, filed Sep. 10, 2015, which is theU.S. National Stage of International Application No. PCT/US2014/023443,filed Mar. 11, 2014, which claims the benefit of U.S. ProvisionalApplication No. 61/792,864, filed Mar. 15, 2013, each of which isincorporated by reference herein for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Contract No.DE-AC02-05CH11231 awarded by the U.S. Department of Energy. Thegovernment has certain rights in this invention.

REFERENCE TO A “SEQUENCE LISTING” SUBMITTED AS AN ASCII TEXT FILE VIAEFS-WEB

This application contains a Sequence Listing file named077429_011630US_SL.TXT, created on Jan. 25, 2021 and containing 158,559bytes, which has been filed electronically in ASCII format. The materialcontained in this text file is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Plant lignocellulosic biomass is used as a renewable feedstock forbiofuel production and is a promising alternative to fossil fuelconsumption. However, a major bottleneck in biofuel production is thequality of available feedstocks. Available feedstocks have a highresistance (recalcitrance) to being reduced into simple sugars that canin turn be converted into fuel. Therefore, improving the compositionand/or digestibility of the raw biomass will have an importantbeneficial impact on lignocellulosic biofuels production.

Lignocellulosic biomass is mainly composed of secondary cell walls,which comprise polysaccharide polymers embedded in lignin. The embeddingof the polysaccharide polymers in lignin reduces their extractabilityand accessibility to hydrolytic enzymes, resulting in cell wallrecalcitrance to enzymatic hydrolysis. Lignin content andsaccharification efficiency of plant cell wall usually are highlynegatively correlated. See, e.g., Chen and Dixon, Nat. Biotechnol.25:759-761 (2007); Jorgensen et al., Biofuel Bioprod. Bior. 1:119-134(2007); and Vinzant et al., Appl. Biochem. Biotechnol. 62:99-104 (1997).However, most attempts at reducing lignin content during plantdevelopment have resulted in severe biomass yield reduction (Franke etal., Plant J. 30:33-45 (2002); Shadle et al., Phytochemistry68:1521-1529 (2007); and Voelker et al., Plant Physiol. 154:874-886(2010)) and therefore, there are few crops having significant ligninreduction. Although silencing strategies have been used to reduce theamount of lignin in plants, there remains a need for methods of reducinglignin in specific cell and tissue types that reduce cell wallrecalcitrance, thus improving the extractability and hydrolysis offermentable sugars from plant biomass.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods of engineering aplant having reduced lignin content. In some embodiments, the methodcomprises:

-   -   introducing into the plant an expression cassette comprising a        polynucleotide that encodes a protein that diverts a monolignol        precursor from a lignin biosynthesis pathway (e.g., a p-coumaryl        alcohol, sinapyl alcohol, and/or coniferyl alcohol biosynthesis        pathway) in the plant, and wherein the polynucleotide is        operably linked to a heterologous promoter; and    -   culturing the plant under conditions in which the protein that        diverts the monolignol precursor from the lignin biosynthesis        pathway is expressed.

In some embodiments, the protein reduces the amount of cytosolic and/orplastidial shikimate that is available for the lignin biosynthesispathway. In some embodiments, the protein is shikimate kinase (AroK),pentafunctional AROM polypeptide (ARO1), dehydroshikimate dehydratase(DsDH), or dehydroshikimate dehydratase (QsuB). In some embodiments, theprotein is substantially identical to an amino acid sequence of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

In some embodiments, the protein reduces the amount of cytosolic and/orplastidial phenylalanine that is available for the lignin biosynthesispathway. In some embodiments, wherein the protein is phenylacetaldehydesynthase (PAAS) or phenylalanine aminomutase (PAM). In some embodiments,the protein is substantially identical to an amino acid sequence of SEQID NO:10 or SEQ ID NO:29.

In some embodiments, the protein reduces the amount of cinnamate and/orcoumarate that is available for the lignin biosynthesis pathway. In someembodiments, the protein is p-coumarate/cinnamatecarboxylmethltransferase (CCMT1) or phenylacrylic acid decarboxylase(PDC). In some embodiments, the protein is substantially identical to anamino acid sequence of SEQ ID NO:12 or SEQ ID NO:30.

In some embodiments, the protein reduces the amount of coumaroyl-CoA,caffeoyl-CoA, and/or feruloyl-CoA that is available for the ligninbiosynthesis pathway. In some embodiments, the protein is2-oxoglutarate-dependent dioxygenase (C2′H), chalcone synthase (CHS),stilbene synthase (SPS), cucuminoid synthase (CUS), or benzalacetone(BAS). In some embodiments, the protein is substantially identical to anamino acid sequence of SEQ ID NO:14, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO;35, or SEQ ID NO: 36.

In some embodiments, the protein activates or potentiates a metabolicpathway that competes with the lignin biosynthesis pathway for the useof monolignol precursors. In some embodiments, the metabolic pathway isa stilbene biosynthesis pathway, a flavonoid biosynthesis pathway, acurcuminoid biosynthesis pathway, or a bensalacetone biosynthesispathway. In some embodiments, the protein is a transcription factor thatactivates or potentiates the flavonoid biosynthesis pathway. In someembodiments, the protein is substantially identical to an amino acidsequence of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45.

In some embodiments, the promoter is a tissue-specific promoter. In someembodiments, the promoter is a secondary cell wall-specific promoter ora fiber cell-specific promoter. In some embodiments, the promoter is anIRX5 promoter. In some embodiments, the promoter is from a gene that isco-expressed in the lignin biosynthesis pathway (phenylpropanoidpathway), e.g., a promoter from a gene expressed in the pathway shown inFIG. 1. In some embodiments, the promoter is a C4H, C3H, HCT, CCR1,CAD4, CAD5, F5H, PAL1, PAL2, 4CL1, or CCoAMT promoter.

In some embodiments, the protein that diverts a monolignol precursorfrom a lignin biosynthesis pathway is targeted to a plastid in theplant. In some embodiments, the polynucleotide comprises a plastidtargeting signal that is substantially identical to the polynucleotidesequence of SEQ ID NO:15.

In some embodiments, the protein diverts a monolignol precursor from asinapyl alcohol and/or coniferyl alcohol biosynthesis pathway. In someembodiments, the plant has reduced content of guaiacyl (G) and syringyl(S) lignin units.

In some embodiments, the plant (or plant part, or seed, flower, leaf, orfruit from the plant) is selected from the group consisting ofArabidopsis, poplar, eucalyptus, rice, corn, switchgrass, sorghum,millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy, barley,turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow, andBrachypodium.

In another aspect, the present invention provides a plant cellcomprising a polynucleotide that encodes a protein that diverts amonolignol precursor from a lignin biosynthesis pathway in the plant,wherein the polynucleotide is operably linked to a heterologouspromoter.

In some embodiments, the plant cell comprises a polynucleotide thatencodes a protein that reduces the amount of cytosolic and/or plastidialshikimate that is available for the lignin biosynthesis pathway. In someembodiments, the protein is shikimate kinase (AroK), pentafunctionalAROM polypeptide (ARO1), dehydroshikimate dehydratase (DsDH), ordehydroshikimate dehydratase (QsuB). In some embodiments, the protein issubstantially identical to an amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, or SEQ ID NO:8.

In some embodiments, the plant cell comprises a polynucleotide thatencodes a protein that reduces the amount of cytosolic and/or plastidialphenylalanine that is available for the lignin biosynthesis pathway. Insome embodiments, wherein the protein is phenylacetaldehyde synthase(PAAS) or phenylalanine aminomutase (PAM). In some embodiments, theprotein is substantially identical to an amino acid sequence of SEQ IDNO:10 or SEQ ID NO:29.

In some embodiments, the plant cell comprises a polynucleotide thatencodes a protein that reduces the amount of cinnamate and/or coumaratethat is available for the lignin biosynthesis pathway. In someembodiments, the protein is p-coumarate/cinnamatecarboxylmethltransferase (CCMT1) or phenylacrylic decarboxylase (PDC).In some embodiments, the protein is substantially identical to an aminoacid sequence of SEQ ID NO:12 or SEQ ID NO:30.

In some embodiments, the plant cell comprises a polynucleotide thatencodes a protein that reduces the amount of coumaroyl-CoA,caffeoyl-CoA, and/or feruloyl-CoA that is available for the ligninbiosynthesis pathway. In some embodiments, the protein is2-oxoglutarate-dependent dioxygenase (C2′H), chalcone synthase (CHS),stilbene synthase (SPS), cucuminoid synthase (CUS), or benzalacetone(BAS). In some embodiments, the protein is substantially identical to anamino acid sequence of SEQ ID NO: 14, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO;35, or SEQ ID NO:36.

In some embodiments, the plant cell comprises a polynucleotide thatencodes a protein activates or potentiates a metabolic pathway thatcompetes with the lignin biosynthesis pathway for the use of monolignolprecursors. In some embodiments, the metabolic pathway is a stilbenebiosynthesis pathway, a flavonoid biosynthesis pathway, a curcuminoidbiosynthesis pathway, or a bensalacetone biosynthesis pathway. In someembodiments, the protein is a transcription factor that activates orpotentiates the flavonoid biosynthesis pathway. In some embodiments, theprotein is substantially identical to an amino acid sequence of SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45.

In some embodiments, the plant cell comprises a tissue-specificpromoter. In some embodiments, the promoter is a secondary cellwall-specific promoter or a fiber cell-specific promoter. In someembodiments, the promoter is an IRX5 promoter. In some embodiments, theplant cell comprises a promoter from a gene that is co-expressed in thelignin biosynthesis pathway (phenylpropanoid pathway), e.g., a promoterfrom a gene expressed in the pathway shown in FIG. 1. In someembodiments, the promoter is a C4H, C3H, HCT, CCR1, CAD4, CAD5, F5H,PAL1, PAL2, 4CL1, or CCoAMT promoter.

In some embodiments, the plant cell comprises a polynucleotide encodinga protein that diverts a monolignol precursor from a lignin biosynthesispathway that is targeted to a plastid in the plant. In some embodiments,the polynucleotide comprises a plastid targeting signal that issubstantially identical to the polynucleotide sequence of SEQ ID NO:15.

In another aspect, the present invention provides plants comprising aplant cell as described herein. In some embodiments, the plant hasreduced lignin content that is substantially localized to secondary cellwall tissue or fiber cells of the plant.

In yet another aspect, the present invention provides methods ofengineering a plant having reduced lignin content by expressing oroverexpressing a competitive inhibitor of a lignin biosynthesis pathwayenzyme. In some embodiments, the method comprises: introducing into theplant an expression cassette comprising a polynucleotide that encodes aprotein that produces a competitive inhibitor of hydroxycinnamoyl-CoAshikimate/quinate hydroxycinnamoyltransferase (HCT) in the plant,wherein the polynucleotide is operably linked to a heterologouspromoter; and culturing the plant under conditions in which the proteinthat produces a competitive inhibitor of HCT is expressed.

In some embodiments, the protein produces one or more of the competitiveinhibitors protocatechuate, gentisate, catechol, 2,3-dihydroxybenzoate,3,6-dihydroxybenzoate, or 3-hydroxy-2-aminobenzoate. In someembodiments, the protein produces the competitive inhibitor of HCTprotocatechuate. In some embodiments, the protein is dehydroshikimatedehydratase (QsuB), dehydroshikimate dehydratase (DsDH), isochorismatesynthase (ICS), salicylic acid 3-hydroxylase (S3H), salicylatehydroxylase (nahG), or salicylate 5-hydroxylase (nagGH).

In some embodiments, the polynucleotide that encodes a protein thatproduces a competitive inhibitor of HCT is operably linked to atissue-specific promoter. In some embodiments, the promoter is asecondary cell wall-specific promoter or a fiber cell-specific promoter.In some embodiments, the promoter is an IRX5 promoter. In someembodiments, the promoter is from a gene that is expressed in the ligninbiosynthesis pathway (phenylpropanoid pathway), e.g., a promoter from agene expressed in the pathway shown in FIG. 1. In some embodiments, thepromoter is a C4H, C3H, HCT, CCR1, CAD4, CAD5, F5H, PAL1, PAL2, 4CL1, orCCoAMT promoter.

In still another aspect, the present invention provides a plant, plantpart, or seed, flower, leaf, or fruit from the plant, or a plant cellcomprising a polynucleotide that encodes a protein that produces acompetitive inhibitor of HCT in the plant, wherein the polynucleotide isoperably linked to a heterologous promoter.

In still another aspect, the present invention provides biomasscomprising plant tissue from a plant or part of a plant as describedherein.

In yet another aspect, the present invention provides methods ofobtaining an increased amount of soluble sugars from a plant in asaccharification reaction. In some embodiments, the method comprisessubjecting a plant as described herein to a saccharification reaction,thereby increasing the amount of soluble sugars that can be obtainedfrom the plant as compared to a wild-type plant.

In still another aspect, the present invention provides methods ofincreasing the digestibility of the biomass for ruminants. In someembodiments, the method comprises introducing an expression cassette asdescribed herein into a plant; culturing the plant under conditions inwhich the protein that diverts the monolignol precursor from the ligninbiosynthesis pathway, or the protein that produces a competitiveinhibitor of HCT, is expressed; and obtaining biomass from the plant,thereby increasing the digestibility of the biomass for ruminants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Representation of the lignin biosynthesis pathway. Modifiedlignin biosynthesis pathway from Fraser and Chapple (2011). Enzymedescriptions: PAL: phenylalanine ammonia-lyase; C4H:cinnamate-4-hydroxylase; 4CL: 4-hydroxycinnamate CoA-ligase; HCT:hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase;C3′H: 4-hydroxycinnamate 3-hydroxylase; CCoAOMT: caffeoyl-CoAO-methyltransferase; CCR: hydroxycinnamoyl-CoA NADPH oxidoreductase;COMT: caffeate O-methyltransferase; CAD: hydroxycinnamyl alcoholdehydrogenase; F5H: ferulate 5-hydroxylase. Name of the ligninprecursors: 1, phenylalanine; 2, cinnamate; 3, p-coumarate; 4,p-coumaroyl-CoA; 5, p-coumaroyl-shikimate/quinate (R=shikimate/quinate);6, caffeoyl-shikimate/quinate; 7, caffeoyl-CoA; 8, feruloyl-CoA; 9,p-coumaraldehyde; 10, coniferaldehyde; 11, 5-hydroxy-coniferaldehyde;12, sinapaldehyde; 13, p-coumaryl alcohol; 14, coniferyl alcohol; 15,sinapyl alcohol.

FIG. 2. Lignin reduction via depletion of shikimate (HCT co-substrate).Strategies for reducing or depleting the amount of shikimate that isavailable for the lignin biosynthesis pathway are shown. (1) The amountof cytosolic shikimate that is available for the lignin biosynthesispathway can be reduced or depleted by expressing a shikimate kinase suchas M. tuberculosis shikimate kinase (“MtAroK”). (2) The amount ofplastidial shikimate that is available for the lignin biosynthesispathway can be reduced or depleted by expressing a pentafunctional aromprotein such as S. cerevisiae pentafunctional arom protein (“ScAro1”).Plastidial expression of the protein can be accomplished via a plastidtargeting signal, e.g., as described herein.

FIG. 3. Lignin reduction via depletion of shikimate and production ofnew stoppers. Strategies for reducing or depleting the amount ofshikimate that is available for the lignin biosynthesis pathway areshown. For example, the amount of plastidial shikimate that is availablefor the lignin biosynthesis pathway can be reduced or depleted byexpressing a dehydroshikimate dehydratase such as C. glutamicumdehydroshikimate dehydratase (“CgQsuB”) or P. anserina dehydroshikimatedehydratase (“PaDsDH”). Plastidial expression of the protein can beaccomplished via a plastid targeting signal, e.g., as described herein.

FIG. 4. Lignin reduction via depletion of phenylalanine (PAL substrate).Strategies for reducing or depleting the amount of phenylalanine that isavailable for the lignin biosynthesis pathway are shown. For example,the amount of (1) cytosolic and/or (2) plastidial phenylalanine that isavailable for the lignin biosynthesis pathway can be reduced or depletedby expressing a phenylacetaldehyde such as P. hybrida phenylacetaldehydesynthase (“PhPAAS”). Plastidial expression of the protein can beaccomplished via a plastid targeting signal, e.g., as described herein.

FIG. 5. Lignin reduction via depletion of phenylalanine (PAL substrate).Strategies for reducing or depleting the amount of phenylalanine that isavailable for the lignin biosynthesis pathway are shown. For example,the amount of (1) cytosolic and/or (2) plastidial phenylalanine that isavailable for the lignin biosynthesis pathway can be reduced or depletedby expressing a phenylalanine aminomutase such as T. canadensisphenylalanine aminomutase (“TcPAM”). Plastidial expression of theprotein can be accomplished via a plastid targeting signal, e.g., asdescribed herein.

FIG. 6. Lignin reduction via depletion of cinnamate (C4H substrate) andcoumarate (4CL substrate). Strategies for reducing or depleting theamount of cinnamate and/or p-coumarate that is available for the ligninbiosynthesis pathway are shown. For example, the amount of cytosoliccinnamate and/or p-coumarate that is available for the ligninbiosynthesis pathway can be reduced or depleted by expressing acinnamate/p-coumarate carboxyl methyltransferase such as O. basilicumcinnamate/p-coumarate carboxyl methyltransferase (“ObCCMT1”).

FIG. 7. Lignin reduction via depletion of cinnamate (C4H substrate) andcoumarate (4CL substrate). Strategies for reducing or depleting theamount of cinnamate and/or p-coumarate that is available for the ligninbiosynthesis pathway are shown. For example, the amount of cytosoliccinnamate and/or p-coumarate that is available for the ligninbiosynthesis pathway can be reduced or depleted by expressing aphenylacrylic decarboxylase (PDC or PAD).

FIG. 8. Lignin reduction via depletion of coumaroyl-CoA (HCT substrate).Strategies for reducing or depleting the amount of coumaroyl-CoA that isavailable for the lignin biosynthesis pathway are shown. For example,the amount of cytosolic coumaroyl-CoA that is available for the ligninbiosynthesis pathway can be reduced or depleted by expressing a2-oxoglutarate-dependent dioxygenase such as R. graveolens C2′H(2-oxoglutarate-dependent dioxygenase) (“RbC2′H”).

FIG. 9. Lignin reduction via depletion of coumaroyl-CoA (HCT substrate).Strategies for reducing or depleting the amount of coumaroyl-CoA that isavailable for the lignin biosynthesis pathway are shown. For example,the amount of cytosolic coumaroyl-CoA that is available for the ligninbiosynthesis pathway can be reduced or depleted by expressing a chalconesynthase (CHS), stilbene synthase (SPS), cucuminoid synthase (CUS), orbenzalacetone (BAS).

FIG. 10. Lignin reduction via depletion of feruloyl-CoA (CCR substrate).Strategies for reducing or depleting the amount of feruloyl-CoA that isavailable for the lignin biosynthesis pathway are shown. For example,the amount of cytosolic feruloyl-CoA that is available for the ligninbiosynthesis pathway can be reduced or depleted by expressing a2-oxoglutarate-dependent dioxygenase such as R. graveolens C2′H(2-oxoglutarate-dependent dioxygenase) (“RbC2′H”).

FIG. 11. Lignin reduction via depletion of caffeoyl-CoA feruloyl-CoA(CCR substrate). Strategies for reducing or depleting the amount ofcaffeoyl-CoA and/or feruloyl-CoA that is available for the ligninbiosynthesis pathway are shown. For example, the amount of cytosoliccaffeoyl-CoA and/or feruloyl-CoA that is available for the ligninbiosynthesis pathway can be reduced or depleted by expressing a chalconesynthase (CHS), synthase (SPS), cucuminoid synthase (CUS), orbenzalacetone (BAS).

FIG. 12. Growth phenotype analysis of S-QsuB lines. Picture of 3weeks-old plants at rosette stage. No phenotypic differences could beobserved between S-QsuB lines and WT plants at the rosette stage.

FIG. 13. Total reducing-sugars released from stem biomass of S-QsuBlines and WT plants after 72 h incubation with a cellulolytic enzymecocktail. Total reducing-sugars released from biomass after hot-waterpretreatment (1 h at 120 C) and incubation with a cellulolytic enzymecocktail (Novozymes Cellic® CTec2) at a loading of 0.88% (g enzyme/gbiomass) were measured using the 3,5-Dinitrosalicylic acid assay asdescribed in Eudes et al. 2012 (Plant Biotech Journal 10(5):609-620).

FIG. 14. Time course for total reducing-sugars released from stembiomass of S-QsuB lines and WT plants after incubation with differentloadings of a cellulolytic enzyme cocktail. Time course for totalreducing-sugars released from biomass after hot-water pretreatment (1 hat 120 C) and incubation with different loadings (0.88%, 0.176% or0.088%; g of enzyme/g of biomass) of a cellulolytic enzyme cocktail(Novozymes Cellic® CTec2). Measurements were performed as described in(Eudes et al. 2012 Plant Biotech Journal 10(5):609-620).

FIG. 15. Total reducing-sugars released from stem biomass of S-DsDHlines after 72 h incubation with a cellulolytic enzyme cocktail. Timecourse for total reducing-sugar released from biomass after hot-waterpretreatment (1 h at 120 C) and incubation with a cellulolytic enzymecocktail (Novozymes Cellic® CTec2) at a loading of 0.88% (g enzyme/gbiomass). Measurements were performed as described in (Eudes et al. 2012Plant Biotech Journal 10(5):609-620).

FIG. 16. QsuB expression in Arabidopsis stems. Detection by Western blotof QsuB tagged with the AttB2 peptide (approximate size 70 kDa) usingthe “universal antibody” and stem proteins from nine independent6-wk-old pC4H::schl::qsuB (C4H::qsuB) T2 transformants. A stem proteinextract from wild type was used as a negative control (WT) and a Ponceaustaining of Rubisco large subunit (rbcL) is shown as a loading control.

FIG. 17. Partial short-range ¹³C-¹H (HSQC) spectra (aromatic region) ofcell-wall material from mature senesced stems of wild-type (WT),pC4H::schl::qsuB-1 (C4H::qsuB-1) and pC4H::schl::qsuB-9 (C4H::qsuB-9)plants. Lignin monomer ratios are provided on the figures.

FIG. 18. Polydispersity of cellulolytic enzyme lignins from wild-typeand C4H::qsuB lines. Cellulolytic enzyme lignins were purified frommature senesced stems of wild-type (WT, black line), pC4H::schl::qsuB-1(C4H::qsuB-1, red line) and pC4H::schl::qsuB-9 (C4H::qsuB-9, purpleline) plants and analyzed for polydispersity by size-exclusionchromatography (SEC). SEC chromatograms were obtained using UV-Ffluorescence (Ex₂₅₀/Em₄₅₀). m, molecular weight.

FIG. 19A-B. Saccharification of biomass from mature senesced stems ofwild-type (WT) and pC4H::schl::qsuB (C4H::qsuB) lines. (A) Amounts ofsugars released from biomass after various pretreatments and 72-henzymatic digestion with cellulase (1% w/w). Values are means±SE of fourbiological replicates (n=4). Asterisks indicate significant differencesfrom the wild type using the unpaired Student's t-test (*P<0.05;**P<0.005). (B) Amounts of sugars released from biomass after hot waterpretreatment and 72-h enzymatic digestion using two different cellulaseloadings (1% or 0.2% w/w). Values are means±SE of four biologicalreplicates (n=4). Asterisks indicate significant differences from thewild type at 1% cellulase loading using the unpaired Student's t-test(*P<0.05; **P<0.005).

FIG. 20. The lignin biosynthetic pathway. Abbreviations: DAHPS,3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; DHQS,3-dehydroquinate synthase; DHQD/SD, 3-dehydroquinate dehydratase; SK,shikimate kinase; ESPS, 3-phosphoshikimate 1-carboxyvinyltransferase;CS, chorismate synthase; CM, chorismate mutase; PAT, prephenateaminotransferase; ADT, arogenate dehydratase; PAL, phenylalanineammonia-lyase; C4H, cinnamate 4-hydroxylase; CSE, caffeoyl shikimateesterase; 4CL, 4-coumarate CoA ligase; CAD, cinnamyl alcoholdehydrogenase; F5H, ferulate 5-hydroxylase; C3H, coumarate3-hydroxylase; COMT, caffeic acid 3-O-methyltransferase; CCR,cinnamoyl-CoA reductase; HCT, hydroxycinnamoyl-Coenzyme Ashikimate/quinate hydroxycinnamoyltransferase; CCoAOMT,caffeoyl/CoA-3-O-methyltransferase; qsuB, 3-dehydroshikimate dehydratasefrom Corynebacterium glutamicum.

FIG. 21. Subcellular localization of SCHL-QsuB. The left panel displaysthe transient expression of SCHL-QsuB-YFP fusion protein expressed underthe control of the 35S promoter in epidermal cells of N. benthamiana andimaged by confocal laser scanning microscopy. The central panel displaysfluorescing chloroplasts and the right panel shows the merged images(colocalizations are visible as yellow dots). Scale bars=20 m.

FIG. 22. Summary of the fold changes observed for the methanol-solublemetabolites extracted from plants expressing QsuB.

FIG. 23. Partial short-range ¹³C-¹H (HSQC) spectra (aliphatic region) ofcell wall material from mature senesced stems of wild-type (WT),pC4H::schl::qsuB-1 (C4H::qsuB-1) and pC4H::schl::qsuB-9 (C4H::qsuB-9)plants.

FIG. 24. Lignin staining by phloroglucinol-HCl of stem sections from5-wk-old wild-type (WT) and pC4H::schl::qsuB (C4H::qsuB) plants.

FIG. 25A-F. LC-MS chromatograms from AtHCT in-vivo activity assays.LC-MS chromatograms of coumarate conjugates produced by AtHCT afterfeeding a recombinant yeast strain co-expressing At4CL5 and AtHCT withp-coumarate and (A) shikimate, (B) 3,6-dihydroxybenzoate, (C)3-hydroxy-2-amino benzoate, (D) 2,3-dihydroxybenzoate, (E) catechol, or(F) protocatechuate are presented. Structures ofcoumarate-dihydroxybenzoate esters are arbitrary shown with an esterlinkage at the 3-hydroxy position of the dihydroxybenzoate ring. Thestructure of coumaroyl-3-hydroxyanthranilate (C) is represented asdetermined in Moglia et al. (34).

FIG. 26. LC-MS chromatogram of p-coumaraldehyde detected inmethanol-soluble extracts of stems from lines expressing QsuB.

FIG. 27. Competitive inhibitor pathways.

FIG. 28. Characteristics and relative molar abundances (%) of thecompounds released after pyro-GC/MS of extractive-free senesced maturestems from wild-type (WT) and pC4H::schl::qsuB (C4H::qsuB) plants.Values in brackets are the SE from duplicate analyses. nd, not detected.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, the term “lignin biosynthesis pathway” refers to anenzymatic pathway (the phenylpropanoid pathway) in plants in which thelignin monomers (p-coumaryl (4-hydroxycinnamyl) alcohol, coniferyl(3-methoxy 4-hydroxycinnamyl) alcohol, and sinapyl (3,5-dimethoxy4-hydroxycinnamyl) alcohol) are synthesized from phenylalanine. Thelignin biosynthesis pathway and enzymatic components of the pathway aredepicted, for example, in FIG. 1.

As used herein, the term “monolignol precursor” refers to a substrate ofthe lignin biosynthesis pathway that is directly or indirectlysynthesized into a lignin monomer. In some embodiments, a monolignolprecursor is a substrate of the lignin biosynthesis pathway that isidentified in any of FIGS. 1-11.

As used herein, the term “protein that diverts a monolignol precursorfrom a lignin biosynthesis pathway” refers to a protein that activates,promotes, potentiates, or enhances expression of an enzymatic reactionor metabolic pathway that decreases the amount of monolignol precursorthat is available for the synthesis of a lignin monomer. The termincludes polymorphic variants, alleles, mutants, and interspecieshomologs to the specific proteins (e.g., enzymes) described herein. Anucleic acid that encodes a protein that diverts a monolignol precursorfrom a lignin biosynthesis pathway (or a nucleic acid that encodes aprotein that diverts a monolignol precursor from a p-coumaryl alcohol,sinapyl alcohol, and/or coniferyl alcohol pathway) refers to a gene,pre-mRNA, mRNA, and the like, including nucleic acids encodingpolymorphic variants, alleles, mutants, and interspecies homologs of theparticular proteins (e.g., enzymes) described herein. In someembodiments, a nucleic acid that encodes a protein that diverts amonolignol precursor from a lignin biosynthesis pathway (1) has anucleic acid sequence that has greater than about 50% nucleotidesequence identity, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher nucleotidesequence identity, preferably over a region of at least about 10, 15,20, 25, 50, 100, 200, 500 or more nucleotides or over the length of theentire polynucleotide, to a nucleic acid sequence of any of SEQ IDNOs:1, 3, 5, 7, 9, 11, or 13; or (2) encodes a polypeptide having anamino acid sequence that has greater than about 50% amino acid sequenceidentity, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequenceidentity, preferably over a region of at least about 25, 50, 100, 200 ormore amino acids or over the length of the entire polypeptide, to apolypeptide encoded by a nucleic acid sequence of any of SEQ ID NOs:1,3, 5, 7, 9, 11, or 13, or to an amino acid sequence of any of SEQ IDNOs:2, 4, 6, 8, 10, 12, 14, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 42, 43, 44, or 45. In some embodiments, a protein thatdiverts a monolignol precursor from a lignin biosynthesis pathway has anamino acid sequence having greater than about 50% amino acid sequenceidentity, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequenceidentity, preferably over a region of at least about 25, 50, 100, 200 ormore amino acids or over the length of the entire polypeptide, to anamino acid sequence of any of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 42, 43, 44, or 45.

The term “protein that produces a competitive inhibitor of HCT” refersto a protein that directly or indirectly produces a molecule that cancompete with p-coumaroyl-CoA and/or shikimate as a substrate forhydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase(HCT), thereby acting as a competitive inhibitor of HCT. Non-limitingexamples of molecules (e.g., metabolites) that can act as competitiveinhibitors of HCT are shown in FIG. 27. In some embodiments, thecompetitive inhibitor of HCT is protocatechuate, catechol,3,6-dihydroxybenzoate, 3-hydroxy-2-aminobenzoate, or2,3-dihydroxybenzoate. Thus, in some embodiments, the protein thatproduces a competitive inhibitor of HCT is a protein (e.g., an enzyme)that directly or indirectly produces protocatechuate, catechol,3,6-dihydroxybenzoate, 3-hydroxy-2-aminobenzoate, or2,3-dihydroxybenzoate, including but not limited to the enzymesdehydroshikimate dehydratase (QsuB), dehydroshikimate dehydratase(DsDH), isochorismate synthase (ICS), salicylic acid 3-hydroxylase(S3H), salicylate hydroxylase (nahG), and salicylate 5-hydroxylase(nagGH). In some embodiments, an in vivo enzymatic assay, for example asdescribed in the Examples section below, can be used to determinewhether a molecule can compete with p-coumaroyl-CoA and/or shikimate asa substrate for HCT.

The terms “polynucleotide” and “nucleic acid” are used interchangeablyand refer to a single or double-stranded polymer of deoxyribonucleotideor ribonucleotide bases read from the 5′ to the 3′ end. A nucleic acidof the present invention will generally contain phosphodiester bonds,although in some cases, nucleic acid analogs may be used that may havealternate backbones, comprising, e.g., phosphoramidate,phosphorothioate, phosphorodithioate, or O-methylphophoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press); positive backbones; non-ionicbackbones, and non-ribose backbones. Thus, nucleic acids orpolynucleotides may also include modified nucleotides that permitcorrect read-through by a polymerase. “Polynucleotide sequence” or“nucleic acid sequence” includes both the sense and antisense strands ofa nucleic acid as either individual single strands or in a duplex. Aswill be appreciated by those in the art, the depiction of a singlestrand also defines the sequence of the complementary strand; thus thesequences described herein also provide the complement of the sequence.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences, as well as the sequenceexplicitly indicated. The nucleic acid may be DNA, both genomic andcDNA, RNA or a hybrid, where the nucleic acid may contain combinationsof deoxyribo- and ribo-nucleotides, and combinations of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xanthinehypoxanthine, isocytosine, isoguanine, etc.

The term “substantially identical,” used in the context of two nucleicacids or polypeptides, refers to a sequence that has at least 50%sequence identity with a reference sequence. Percent identity can be anyinteger from 50% to 100%. Some embodiments include at least: 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%, compared to a reference sequence using the programsdescribed herein; preferably BLAST using standard parameters, asdescribed below. For example, a first polynucleotide is substantiallyidentical to a second polynucleotide sequence if the firstpolynucleotide sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe second polynucleotide sequence.

Two nucleic acid sequences or polypeptide sequences are said to be“identical” if the sequence of nucleotides or amino acid residues,respectively, in the two sequences is the same when aligned for maximumcorrespondence as described below. The terms “identical” or percent“identity,” in the context of two or more nucleic acids or polypeptidesequences, refer to two or more sequences or subsequences that are thesame or have a specified percentage of amino acid residues ornucleotides that are the same, when compared and aligned for maximumcorrespondence over a comparison window, as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. When percentage of sequence identity is used inreference to proteins or peptides, it is recognized that residuepositions that are not identical often differ by conservative amino acidsubstitutions, where amino acids residues are substituted for otheramino acid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. Where sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well known to those of skill in the art. Typically thisinvolves scoring a conservative substitution as a partial rather than afull mismatch, thereby increasing the percentage sequence identity.Thus, for example, where an identical amino acid is given a score of 1and a non-conservative substitution is given a score of zero, aconservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated according to, e.g.,the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17(1988) e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection.

Algorithms that are suitable for determining percent sequence identityand sequence similarity are the BLAST and BLAST 2.0 algorithms, whichare described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 andAltschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (NCBI) web site. Thealgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al,supra). These initial neighborhood word hits acts as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a word size (W) of28, an expectation (E) of 10, M=1, N=−2, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults aword size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)).

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.01, more preferably lessthan about 10⁻⁵, and most preferably less than about 10⁻²⁰.

Nucleic acid or protein sequences that are substantially identical to areference sequence include “conservatively modified variants.” Withrespect to particular nucleic acid sequences, conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that eachcodon in a nucleic acid (except AUG, which is ordinarily the only codonfor methionine) can be modified to yield a functionally identicalmolecule. Accordingly, each silent variation of a nucleic acid whichencodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, in a nucleic acid, peptide, polypeptide, or proteinsequence which alters a single amino acid or a small percentage of aminoacids in the encoded sequence is a “conservatively modified variant”where the alteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(see, e.g., Creighton, Proteins (1984)).

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other, or a third nucleic acid,under stringent conditions. Stringent conditions are sequence dependentand will be different in different circumstances. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe.Typically, stringent conditions will be those in which the saltconcentration is about 0.02 molar at pH 7 and the temperature is atleast about 60° C. For example, stringent conditions for hybridization,such as RNA-DNA hybridizations in a blotting technique are those whichinclude at least one wash in 0.2×SSC at 55° C. for 20 minutes, orequivalent conditions.

As used herein, the term “promoter” refers to a polynucleotide sequencecapable of driving transcription of a DNA sequence in a cell. Thus,promoters used in the polynucleotide constructs of the invention includecis- and trans-acting transcriptional control elements and regulatorysequences that are involved in regulating or modulating the timingand/or rate of transcription of a gene. For example, a promoter can be acis-acting transcriptional control element, including an enhancer, apromoter, a transcription terminator, an origin of replication, achromosomal integration sequence, 5′ and 3′ untranslated regions, or anintronic sequence, which are involved in transcriptional regulation.These cis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.) genetranscription. Promoters are located 5′ to the transcribed gene, and asused herein, include the sequence 5′ from the translation start codon(i.e., including the 5′ untranslated region of the mRNA, typicallycomprising 100-200 bp). Most often the core promoter sequences liewithin 1-5 kb of the translation start site, more often within 1 kbp andoften within 500 bp of the translation start site. By convention, thepromoter sequence is usually provided as the sequence on the codingstrand of the gene it controls.

A “constitutive promoter” is one that is capable of initiatingtranscription in nearly all cell types, whereas a “cell type-specificpromoter” initiates transcription only in one or a few particular celltypes or groups of cells forming a tissue. In some embodiments, thepromoter is secondary cell wall-specific and/or fiber cell-specific. A“fiber cell-specific promoter” refers to a promoter that initiatessubstantially higher levels of transcription in fiber cells as comparedto other non-fiber cells of the plant. A “secondary cell wall-specificpromoter” refers to a promoter that initiates substantially higherlevels of transcription in cell types that have secondary cell walls,e.g., lignified tissues such as vessels and fibers, which may be foundin wood and bark cells of a tree, as well as other parts of plants suchas the leaf stalk. In some embodiments, a promoter is fibercell-specific or secondary cell wall-specific if the transcriptionlevels initiated by the promoter in fiber cells or secondary cell walls,respectively, are at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold higheror more as compared to the transcription levels initiated by thepromoter in other tissues, resulting in the encoded proteinsubstantially localized in plant cells that possess fiber cells orsecondary cell wall, e.g., the stem of a plant. Non-limiting examples offiber cell and/or secondary cell wall specific promoters include thepromoters directing expression of the genes IRX1, IRX3, IRX5, IRX7,IRX8, IRX9, IRX10, IRX14, NST1, NST2, NST3, MYB46, MYB58, MYB63, MYB83,MYB85, MYB103, PAL1, PAL2, C3H, CcOAMT, CCR1, F5H, LAC4, LAC17, CADc,and CADd. See, e.g., Turner et al 1997; Meyer et al 1998; Jones et al2001; Franke et al 2002; Ha et al 2002;Rohde et al 2004; Chen et al2005; Stobout et al 2005; Brown et al 2005; Mitsuda et al 2005; Zhong etal 2006; Mitsuda et al 2007; Zhong et al 2007a, 2007b; Zhou et al 2009;Brown et al 2009; McCarthy et al 2009; Ko et al 2009; Wu et al 2010;Berthet et al 2011. In some embodiments, a promoter is substantiallyidentical to a promoter from the lignin biosynthesis pathway (e.g., apromoter for a gene encoding a protein shown in FIG. 1). Non-limitingexamples of promoter sequences are provided herein as SEQ ID NOs:17-28.A promoter originated from one plant species may be used to direct geneexpression in another plant species.

A polynucleotide is “heterologous” to an organism or a secondpolynucleotide sequence if it originates from a foreign species, or, iffrom the same species, is modified from its original form. For example,when a polynucleotide encoding a polypeptide sequence is said to beoperably linked to a heterologous promoter, it means that thepolynucleotide coding sequence encoding the polypeptide is derived fromone species whereas the promoter sequence is derived from another,different species; or, if both are derived from the same species, thecoding sequence is not naturally associated with the promoter (e.g., isa genetically engineered coding sequence, e.g., from a different gene inthe same species, or an allele from a different ecotype or variety, or agene that is not naturally expressed in the target tissue).

The term “operably linked” refers to a functional relationship betweentwo or more polynucleotide (e.g., DNA) segments. Typically, it refers tothe functional relationship of a transcriptional regulatory sequence toa transcribed sequence. For example, a promoter or enhancer sequence isoperably linked to a DNA or RNA sequence if it stimulates or modulatesthe transcription of the DNA or RNA sequence in an appropriate host cellor other expression system. Generally, promoter transcriptionalregulatory sequences that are operably linked to a transcribed sequenceare physically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory sequences, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

The term “expression cassette” refers to a nucleic acid construct that,when introduced into a host cell, results in transcription and/ortranslation of an RNA or polypeptide, respectively. Antisense or senseconstructs that are not or cannot be translated are expressly includedby this definition. In the case of both expression of transgenes andsuppression of endogenous genes (e.g., by antisense, RNAi, or sensesuppression) one of skill will recognize that the insertedpolynucleotide sequence need not be identical, but may be onlysubstantially identical to a sequence of the gene from which it wasderived. As explained herein, these substantially identical variants arespecifically covered by reference to a specific nucleic acid sequence.

The term “plant,” as used herein, refers to whole plants and includesplants of a variety of a ploidy levels, including aneuploid, polyploid,diploid, and haploid. The term “plant part,” as used herein, refers toshoot vegetative organs and/or structures (e.g., leaves, stems andtubers), branches, roots, flowers and floral organs (e.g., bracts,sepals, petals, stamens, carpels, anthers), ovules (including egg andcentral cells), seed (including zygote, embryo, endosperm, and seedcoat), fruit (e.g., the mature ovary), seedlings, and plant tissue(e.g., vascular tissue, ground tissue, and the like), as well asindividual plant cells, groups of plant cells (e.g., cultured plantcells), protoplasts, plant extracts, and seeds. The class of plants thatcan be used in the methods of the invention is generally as broad as theclass of higher and lower plants amenable to transformation techniques,including angiosperms (monocotyledonous and dicotyledonous plants),gymnosperms, ferns, and multicellular algae.

The term “biomass,” as used herein, refers to plant material that isprocessed to provide a product, e.g., a biofuel such as ethanol, orlivestock feed, or a cellulose for paper and pulp industry products.Such plant material can include whole plants, or parts of plants, e.g.,stems, leaves, branches, shoots, roots, tubers, and the like.

The term “reduced lignin content” encompasses reduced amount of ligninpolymer, reduced amount of either or both of the guaiacyl (G) and/orsyringyl (S) lignin units, reduced size of a lignin polymer, e.g., ashorter lignin polymer chain due to a smaller number of monolignolsbeing incorporated into the polymer, a reduced degree of branching ofthe lignin polymer, or a reduced space filling (also called a reducedpervaded volume). In some embodiments, a reduced lignin polymer can beshown by detecting a decrease in the molecular weight of the polymer ora decrease in the number of monolignols by at least 2%, 5%, 10%, 20%,25%, 30%, 40%, 50%, or more, when compared to the average ligninmolecule in a control plant (e.g., a non-transgenic plant). In someembodiments, reduced lignin content can be shown by detecting a decreasein the number or amount of guaiacyl (G) and/or syringyl (S) lignin unitsin the plant as compared to a control plant (e.g., a non-transgenicplant). In some embodiments, a plant as described herein has reducedlignin content if the amount of guaiacyl (G) and/or syringyl (S) ligninunits in the plant is decreased by at least about 2%, 5%, 10%, 20%, 25%,30%, 40%, 50% or more, as compared to a control plant. Methods fordetecting reduced lignin content are described in detail below.

II. Introduction

Plant cell walls constitute a polysaccharidic network of cellulosemicrofibrils and hemicellulose embedded in an aromatic polymer known aslignin. This ramified polymer is mainly composed of threephenylpropanoid-derived phenolics (i.e., monolignols) namedp-coumaryl,coniferyl, and sinapyl alcohols which represent thep-hydroxyphenyl (H),guaiacyl (G) and syringyl (S) lignin units (Boerjan et al., 2003).Monolignols have a C₆C₃ carbon skeleton which consists of a phenyl ring(C₆) and a propane (C₃) side chain. Lignin is crucial for thedevelopment of terrestrial plants as it confers recalcitrance to plantcell walls. It also provides mechanical strength for upright growth,confers hydrophobicity to vessels that transport water, and acts as aphysical barrier against pathogens that degrade cell walls (Boudet,2007). Notably, lignin content and composition are finely regulated inresponse to environmental biotic and abiotic stresses (Moura et al.,2010).

Economically, lignocellulosic biomass from plant cell walls is widelyused as raw material for the production of pulp in paper industry and asruminant livestock feed. Plant feedstocks also represent a source offermentable sugars for the production of synthetic molecules such aspharmaceuticals and transportation fuels using engineered microorganisms(Keasling, 2010). However, negative correlations exist between lignincontent in plant biomass and pulp yield, forage digestibility, orpolysaccharides enzymatic hydrolysis (de Vrije et al., 2002; Reddy etal., 2005; Dien et al., 2006; Chen and Dixon, 2007; Dien et al., 2009;Taboada et al., 2010; Elissetche et al., 2011; Studer et al., 2011).Consequently, reducing lignin recalcitrance in plant feedstocks is amajor focus of interest, especially in the lignocellulosic biofuelsfield for which efficient enzymatic conversion of polysaccharides intomonosaccharides is crucial to achieve economically and environmentallysustainable production (Carroll and Somerville, 2009).

Lignin biosynthesis is well characterized and well conserved across landplants (Weng and Chapple 2010). Genetic modifications such as silencingof genes involved in particular steps of this pathway or its regulationhave been employed to reduce lignin content (Simmons et al., 2010;Umezawa, 2010) but this approach often results in undesired phenotypessuch as dwarfism, sterility, reduction of plant biomass, and increasedsusceptibly to environmental stress and pathogens (Bonawitz and Chapple,2010). These pleiotropic effects are generally the consequences of aloss of secondary cell wall integrity, accumulation of toxicintermediates, constitutive activation of defense responses, ordepletion of other phenylpropanoid-derived metabolites which areessential for plant development and defense (Li et al., 2008; Naoumkinaet al., 2010, Gallego-Giraldo et al., 2011). Alternatively, changing therecalcitrant structure and physico-chemical properties of lignin can beachieved by modifying its monomer composition. For example,incorporation of coniferyl ferulate into lignin improves enzymaticdegradation of cell wall polysaccharides (Grabber et al., 2008).Recently, it has been demonstrated that enrichment in 5-hydroxy-G unitsand reduction in S units in lignin contribute to enhancedsaccharification efficiencies without affecting drastically biomassyields and lignin content (Weng et al., 2010; Dien et al., 2011; Fu etal., 2011).

The present invention provides an alternative strategy to reduce lignincontent (e.g., reducing the amount of p-hydroxyphenyl (H), guaiacyl (G)and/or syringyl (S) lignin units, or any combination of H-lignin,G-lignin, and S-lignin units). In this strategy, the plant is engineeredto express one or more proteins that diverts or shunts a monolignolprecursor from a lignin biosynthesis pathway (e.g., a p-coumarylalcohol, sinapyl alcohol, and/or coniferyl alcohol biosynthesis pathway)into a competitive pathway. By diverting or shunting the production ofmonolignol precursors fromp-hydroxyphenyl (H), guaiacyl (G) and/orsyringyl (S) lignin unit production to the production of alternativeproducts (e.g., stilbenes, flavonoids, curcuminoids, or bensalacetones,protocatechuates, aromatic amino acids, vitamins, quinones, or volatilecompounds) as described herein, the amount of lignin content or itscomposition, e.g., in specific cell or tissue types such as in secondarycell wall, can be altered in order to enhance saccharificationefficiencies without dramatically affecting biomass yield. The presentinvention also provides plants that are engineered by the methoddescribed herein, as well as a plant cell from such a plant, a seed,flower, leaf, or fruit from such a plant, a plant cell that contains anexpression cassette described herein for expressing a protein diverts orshunts a monolignol precursor from a lignin biosynthesis pathway into acompetitive pathway, and biomass comprising plant tissue from the plantor part of the plant described herein.

III. Plants Having Reduced Lignin Content

A. Expression of a Protein that Diverts a Monolignol Precursor from aLignin Biosynthesis Pathway

In one aspect, the present invention provides a method of engineering aplant having reduced lignin content (e.g., reduced amount of ligninpolymers, reduced size of lignin polymers, reduced degree of branchingof lignin polymers, or reduced space filling). In some embodiments, theplant has reduced lignin content that is substantially localized tospecific cell and/or tissue types in the plant. For example, in someembodiments the plant has reduced lignin content that is substantiallylocalized to secondary cell walls and/or fiber cells. In someembodiments, the method comprises:

-   -   introducing into the plant an expression cassette comprising a        polynucleotide that encodes a protein that diverts a monolignol        precursor from a lignin biosynthesis pathway (e.g., a p-coumaryl        alcohol, sinapyl alcohol, and/or coniferyl alcohol biosynthesis        pathway) in the plant, and wherein the polynucleotide is        operably linked to a heterologous tissue-specific promoter; and    -   culturing the plant under conditions in which the protein that        diverts the monolignol precursor from the lignin biosynthesis        pathway (e.g., the p-coumaryl alcohol, sinapyl alcohol, or        coniferyl alcohol biosynthesis pathway) is expressed.

In some embodiments, the gene that encodes a protein that diverts amonolignol precursor from a lignin biosynthesis pathway (e.g., ap-coumaryl alcohol, sinapyl alcohol, and/or coniferyl alcoholbiosynthesis pathway) reduces the amount of cytosolic and/or plastidialshikimate that is available for the p-coumaryl alcohol, sinapyl alcohol,or coniferyl alcohol biosynthesis pathway; reduces the amount ofcytosolic and/or plastidial phenylalanine that is available for thep-coumaryl alcohol, sinapyl alcohol, or coniferyl alcohol biosynthesispathway; reduces the amount of cinnamate and/or coumarate that isavailable for the p-coumaryl alcohol, sinapyl alcohol, or coniferylalcohol biosynthesis pathway; and/or reduces the amount ofcoumaroyl-CoA, caffeoyl-CoA, and/or feruloyl-CoA that is available forthe p-coumaryl alcohol, sinapyl alcohol, or coniferyl alcoholbiosynthesis pathway. In some embodiments, the gene that encodes aprotein that diverts a monolignol precursor from a lignin biosynthesispathway (e.g., a p-coumaryl alcohol, sinapyl alcohol, and/or coniferylalcohol biosynthesis pathway) activates or potentiates a metabolicpathway that competes with the p-coumaryl alcohol, sinapyl alcohol, orconiferyl alcohol biosynthesis pathway biosynthesis pathway for the useof monolignol precursors, including but not limited to a metabolicpathway selected from a stilbene biosynthesis pathway, a flavonoidbiosynthesis pathway, and an anthocyanin biosynthesis pathway.

An expression cassette as described herein, when introduced into aplant, results in the plant having reduced lignin content (e.g., reducedamount of lignin polymers, reduced size of lignin polymers, reduceddegree of branching of lignin polymers, or reduced space filling) thatis specifically localized to certain cell and/or tissue types (e.g.,specifically localized to secondary cell walls and/or fiber cells), thusreducing cell wall recalcitrance to enzymatic hydrolysis while avoidingdefects in plant growth or reductions in biomass yield.

One of skill in the art will understand that the protein that diverts amonolignol precursor from a lignin biosynthesis pathway that isintroduced into the plant by an expression cassette described hereindoes not have to be identical to the protein sequences described herein(e.g., the protein sequences of SEQ ID NOs:2, 4, 6, 8, 10, 12, or 14).In some embodiments, the protein that is introduced into the plant by anexpression cassette is substantially identical (e.g., at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical) to a protein sequence describedherein (e.g., a protein sequence of SEQ ID NOs:2, 4, 6, 8, 10, 12, or14). In some embodiments, the protein that is introduced into the plantby an expression cassette is a homolog, ortholog, or paralog of aprotein that diverts a monolignol precursor from a lignin biosynthesispathway as described herein (e.g., a protein sequence of SEQ ID NOs:2,4, 6, 8, 10, 12, or 14).

Gene and protein sequences for enzymes that divert a monolignolprecursor from a lignin biosynthesis pathway are described in theSequence Listing herein. Additionally, gene and protein sequences forthese proteins, and methods for obtaining the genes or proteins, areknown and described in the art. One of skill in the art will recognizethat these gene or protein sequences known in the art and/or asdescribed herein can be modified to make substantially identicalenzymes, e.g., by making conservative substitutions at one or more aminoacid residues. One of skill will also recognize that the known sequencesprovide guidance as to what amino acids may be varied to make asubstantially identical enzyme. For example, using an amino acidsequence alignment between two or more protein sequences, one of skillwill recognize which amino acid residues are not highly conserved andthus can likely be changed without resulting in a significant effect onthe function of the enzyme.

Proteins that Reduce the Amount of Shikimate

In some embodiments, a protein that diverts a monolignol precursor froma lignin biosynthesis pathway reduces the amount of cytosolic and/orplastidial shikimate that is available for the lignin biosynthesispathway. Examples of such a protein are shown in FIGS. 2 and 3. In someembodiments, the protein is an enzyme that modifies a shikimatesubstrate, e.g., a shikimate kinase or a pentafunctional arom protein.In some embodiments, the protein is an enzyme that utilizes shikimate inthe synthesis of another compound (e.g., a protocatechuate, an aromaticamino acid, a vitamin, or a quinone), e.g., a dehydroshikimatedehydratase.

Non-limiting examples of a shikimate kinase enzyme are described in Guet al., J. Mol. Biol. 319:779-789 (2002). In some embodiments, theprotein is a Mycobacterium tuberculosis shikimate kinase (AroK) havingthe amino acid sequence set forth in SEQ ID NO:2. In some embodiments,the protein is substantially identical (e.g., at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical) to the amino acid sequence of SEQ IDNO:2. In some embodiments, the protein is a homolog of a Mycobacteriumtuberculosis shikimate kinase (AroK) having the amino acid sequence setforth in SEQ ID NO:2. In some embodiments, a polynucleotide encoding theshikimate kinase comprises a polynucleotide sequence that is identicalor substantially identical (e.g., at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical) to SEQ ID NO:1.

Non-limiting examples of a pentafunctional arom protein are described inDuncan et al., Biochem. J. 246:375-386 (1987). In some embodiments, theprotein is a Saccharomyces cerevisiae pentafunctional arom enzyme (Aro1)having the amino acid sequence set forth in SEQ ID NO:4. In someembodiments, the protein is substantially identical (e.g., at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical) to the amino acid sequence of SEQID NO:4. In some embodiments, the protein is a homolog of aSaccharomyces cerevisiae pentafunctional arom enzyme (Aro1) having theamino acid sequence set forth in SEQ ID NO:4. In some embodiments, apolynucleotide encoding the pentafunctional arom protein comprises apolynucleotide sequence that is identical or substantially identical(e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical) to SEQ IDNO:3.

Non-limiting examples of a dehydroshikimate dehydratase are described inTeramoto et al., Appl. Environ. Microbiol. 75:3461-3468 (2009) andHansen et al., Appl. Environ. Microbiol. 75:2765-2774 (2009). In someembodiments, the protein is a Corynebacterium glutamicumdehydroshikimate dehydratase (QsuB) having the amino acid sequence setforth in SEQ ID NO:6 or a Podospora anserina dehydroshikimatedehydratase (DsDH) having the amino acid sequence set forth in SEQ IDNO:8. In some embodiments, the protein is substantially identical (e.g.,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical) to the amino acidsequence of SEQ ID NO:6 or SEQ ID NO:8. In some embodiments, the proteinis a homolog of a Corynebacterium glutamicum dehydroshikimatedehydratase (QsuB) having the amino acid sequence set forth in SEQ IDNO:6 or a homolog of the Podospora anserina dehydroshikimate dehydratase(DsDH) having the amino acid sequence set forth in SEQ ID NO:8. In someembodiments, a polynucleotide encoding the dehydroshikimate dehydratasecomprises a polynucleotide sequence that is identical or substantiallyidentical (e.g., at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical) to SEQID NO:5 or SEQ ID NO:7.

Proteins that Reduce the Amount of Phenylalanine

In some embodiments, a protein that diverts a monolignol precursor froma lignin biosynthesis pathway reduces the amount of cytosolic and/orplastidial phenylalanine that is available for the lignin biosynthesispathway. Examples of such a protein are shown in FIGS. 4 and 5. In someembodiments, the protein is an enzyme that modifies a phenylalaninesubstrate. In some embodiments, the protein is an enzyme that utilizesphenylalanine in the synthesis of another compound (e.g., a volatilecompound), e.g., a phenylacetaldehyde synthase or a phenylalanineaminomutase.

Non-limiting examples of a phenylacetaldehyde synthase are described inKaminaga et al., J. Biol. Chem. 281:23357-23366 (2006) and in Farhi etal., PlantMol. Biol. 72:235-245 (2010). In some embodiments, the proteinis a Petunia hybrida phenylacetaldehyde synthase (PAAS) having the aminoacid sequence set forth in SEQ ID NO: 10. In some embodiments, theprotein is substantially identical (e.g., at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical) to the amino acid sequence of SEQ ID NO:10. In someembodiments, the protein is a homolog of a Petunia hybridaphenylacetaldehyde synthase (PAAS) having the amino acid sequence setforth in SEQ ID NO: 10. In some embodiments, a polynucleotide encodingthe phenylacetaldehyde synthase comprises a polynucleotide sequence thatis identical or substantially identical (e.g., at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical) to SEQ ID NO:9.

Non-limiting examples of a phenylalanine aminomutase are described inFeng et al., Biochemistry 50:2919-2930 (2011). In some embodiments, theprotein is a T. canadensis phenylalanine aminomutase (PAM) having theamino acid sequence set forth in SEQ ID NO:29. In some embodiments, theprotein is substantially identical (e.g., at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical) to the amino acid sequence of SEQ ID NO:29. In someembodiments, the protein is a homolog of a T. canadensis phenylalanineaminomutase (PAM) having the amino acid sequence set forth in SEQ IDNO:29.

Proteins that Reduce the Amount of Cinnamate and/or Coumarate

In some embodiments, a protein that diverts a monolignol precursor froma lignin biosynthesis pathway reduces the amount of cinnamate and/orcoumarate that is available for the lignin biosynthesis pathway.Examples of such a protein are shown in FIGS. 6 and 7. In someembodiments, the protein is an enzyme that modifies a cinnamate and/orcoumarate substrate, e.g., a cinnamate/p-coumarate carboxylmethyltransferase. In some embodiments, the protein is an enzyme thatutilizes cinnamate and/or coumarate in the synthesis of another compound(e.g., a volatile compound, e.g., styrene or p-hydroxystyrene), e.g.,phenylacrylic acid decarboxylase or ferulic acid decarboxylase.

Non-limiting examples of a cinnamate/p-coumarate carboxylmethyltransferase enzyme are described in Kapteyn et al., Plant Cell19:3212-3229 (2007). In some embodiments, the protein is a Ocimumbasilicum cinnamate/p-coumarate carboxyl methyltransferase (CCMT) havingthe amino acid sequence set forth in SEQ ID NO:12. In some embodiments,the protein is substantially identical (e.g., at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical) to the amino acid sequence of SEQ ID NO:12. In some embodiments, the protein is a homolog of a Ocimum basilicumcinnamate/p-coumarate carboxyl methyltransferase (CCMT) having the aminoacid sequence set forth in SEQ ID NO:12. In some embodiments, apolynucleotide encoding the cinnamate/p-coumarate carboxylmethyltransferase comprises a polynucleotide sequence that is identicalor substantially identical (e.g., at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical) to SEQ ID NO: 11.

Non-limiting examples of a phenylacrylic acid decarboxylase aredescribed in McKenna et al., Metab Eng 13:544-554 (2011). In someembodiments, the protein is a P. penosaceus phenylacrylic aicddecarboxylase (PDC) having the amino acid sequence set forth in SEQ IDNO:30. In some embodiments, the protein is substantially identical(e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical) to the aminoacid sequence of SEQ ID NO:30. In some embodiments, the protein is ahomolog of a P. penosaceus phenylacrylic acid decarboxylase (PDC) havingthe amino acid sequence set forth in SEQ ID NO:30.

Proteins that Reduce the Amount of Coumaroyl-CoA, Caffeoyl-CoA, and/orFeruloyl-CoA

In some embodiments, a protein that diverts a monolignol precursor froma lignin biosynthesis pathway reduces the amount of coumaroyl-CoA and/orferuloyl-CoA that is available for the lignin biosynthesis pathway.Examples of such a protein are shown in FIGS. 8-11. In some embodiments,the protein is an enzyme that modifies a coumaroyl-CoA and/orferuloyl-CoA substrate. In some embodiments, the protein is an enzymethat utilizes coumaroyl-CoA and/or feruloyl-CoA in the synthesis ofanother compound (e.g., umbelliferone, a volatile compound, scopoletin,chalcone, trihydroxychalcone, stilbene, curuminoid, or benzylacetone),e.g., 2-oxoglutarase-dependent dioxygenase, chalcone synthase, stilbenesynthase, cucuminoid synthase, or benzalacetone synthase.

A non-limiting example of a 2-oxoglutarase-dependent dioxygenase enzymeis described in Vialart et al., Plant J. 70:460-470 (2012). In someembodiments, the protein is a Ruta graveolens 2-oxoglutarase-dependentdioxygenase (C2′H) having the amino acid sequence set forth in SEQ IDNO: 14. In some embodiments, the protein is substantially identical(e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical) to the aminoacid sequence of SEQ ID NO:14. In some embodiments, the protein is ahomolog of a Ruta graveolens 2-oxoglutarase-dependent dioxygenase (C2′H)having the amino acid sequence set forth in SEQ ID NO: 14. In someembodiments, a polynucleotide encoding the oxoglutarase-dependentdioxygenase comprises a polynucleotide sequence that is identical orsubstantially identical (e.g., at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical) to SEQ ID NO:13.

Other non-limiting examples of proteins that reduce the amount ofcoumaroyl-CoA, caffeoyl-CoA, and/or feruloyl-CoA that is available forthe lignin biosynthesis pathway chalcone synthase (CHS), stilbenesynthase (SPS), cucuminoid synthase (CUS), or benzalacetone synthase(BAS), described in Katsuyama et al., J. Biol. Chem. 282:37702-37709(2007); Sydor et al., Appl. Environ. Microbiol. 76:3361-3363 (2010);Jiang et al., Phytochemistry 67:2531-2540 (2006); Abe and Morita, Nat.Prod. Rep. 27:809 (2010); Dao et al., Phytochem Rev. 10:397-412 (2011);Suh et al., Biochem J. 350:229-235 (2000); Tropf et al., J. Biol. Chem.270:7922-7928 (1995); Knogge et al., Arch. Biochem. Biophys. 250:364-372(1986); Ferrer et al., Nat. Struct. Biol. 6:775-784 (1999); Miyazono etal., Proteins 79:669-673 (2010); and Abe et al., Eur. J. Biochem.268:3354-3359 (2001). In some embodiments, the protein is aPhyscomitrella patens CHS having the amino acid sequence set forth inSEQ ID NO:31; an Arabidopsis thaliana CHS having the amino acid sequenceset forth in SEQ ID NO:32; a Vitis vinifera SPS having the amino acidsequence set forth in SEQ ID NO:33; an Oryza sativa CUS having the aminoacid sequence set forth in SEQ ID NO:34 or SEQ ID NO:35; or a Rheumpalmatum BAS having the amino acid sequence set forth in SEQ ID NO:36;or a homolog thereof. In some embodiments, the protein is substantiallyidentical (e.g., at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical) to theamino acid sequence of any of SEQ ID NOs:31, 32, 33, 34, 35, or 36.

Proteins that Activate a Competitive Metabolic Pathway

In some embodiments, a protein that diverts a monolignol precursor froma lignin biosynthesis pathway activates, upregulates, or potentiates ametabolic pathway that competes with the lignin biosynthesis pathwaybiosynthesis pathway for the use of monolignol precursors. Non-limitingexamples of metabolic pathways that are competitive with the ligninbiosynthesis pathway include the stilbene biosynthesis pathway, theflavonoid biosynthesis pathway, the curcuminoid biosynthesis pathway,and the bensalacetone biosynthesis pathway. Thus, in some embodiments,the protein that diverts a monolignol precursor from a ligninbiosynthesis pathway is a protein (e.g., a transcription factor, aTALE-based artificial transcription factor (see Zhang et al., Nat.Biotechnol. 29:149-153 (2011)), or an enzyme) that activates,upregulates, induces, or potentiates a stilbene biosynthesis pathway, aflavonoid biosynthesis pathway, a curcuminoid biosynthesis pathway, or abensalacetone biosynthesis pathway

As one non-limiting example, a protein can be expressed that activates,upregulates, induces, or potentiates a flavonoid biosynthesis pathway.The flavonoid biosynthesis pathway utilizes monolignol precursors suchas coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA from the ligninbiosynthesis pathway for the synthesis of flavonoids such as chalcones,flavonones, dihydroflavonols, flavonols, and anthocyanins. See FIGS. 9and 11. In some embodiments, the protein that diverts a monolignolprecursor from a lignin biosynthesis pathway is a protein thatactivates, upregulates, induces, or potentiates the expression and/oractivity of an enzyme in the flavonoid biosynthesis pathway (e.g., anenzyme such as chalcone synthase or flavonol synthase). In someembodiments, the protein that diverts a monolignol precursor from alignin biosynthesis pathway is a transcription factor. Transcriptionfactors in the flavonoid biosynthesis pathway are known in the art. See,e.g., Bovy et al., Plant Cell 14:2509-2526 (2002); Tohge et al., PlantJ.42:218-235 (2005); Peel et al., Plant J. 59:136-149 (2009); Pattanaik etal., Planta 231:1061-1076 (2010); and Hichri et al., J Exp Botany62:2465-2483 (2011); incorporated by reference herein. Non-limitingexamples of transcription factors in the flavonoid biosynthesis pathwayinclude MYB transcription factors, basic helix-loop-helix (bHLH)transcription factors, and WD40 transcription factors. In someembodiments, the protein is an Arabidopsis thaliana PAP1 R2R3 MYBtranscription factor having the amino acid sequence set forth in SEQ IDNO:37; an Arabidopsis thaliana PAP2 R2R3 MYB transcription factor havingthe amino acid sequence set forth in SEQ ID NO:38; an Arabidopsisthaliana TT2 R2R3 MYB transcription factor having the amino acidsequence set forth in SEQ ID NO:39; a Nicotiana tabacum NtAn2 R2R3 MYBtranscription factor having the amino acid sequence set forth in SEQ IDNO:40; a Medicago truncatula LAPi R2R3 MYB transcription factor havingthe amino acid sequence set forth in SEQ ID NO:41; a Zea mays MYB-C R2R3transcription factor having the amino acid sequence set forth in SEQ IDNO:42; a Zea mays MYC-Lc BHLH transcription factor having the amino acidsequence set forth in SEQ ID NO:43; an Arabidopsis thaliana TT8 BHLHtranscription factor having the amino acid sequence set forth in SEQ IDNO:44; or a Vitis vinfera Myc1 BHLH transcription factor having theamino acid sequence set forth in SEQ ID NO:45; or a homolog thereof. Insome embodiments, the protein is substantially identical (e.g., at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical) to the amino acid sequenceof any of SEQ ID NOs:37, 38, 39, 40, 41, 42, 43, 44, or 45.

In some embodiments, a plant is engineered to express two, three, fouror more proteins as described herein. In some embodiments, the plantexpresses two or more proteins, each of which is identical orsubstantially identical to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 42, 43, 44, or 45. Insome embodiments, the two or more proteins utilize different substratesor activate different pathways; for example, in some embodiments theplant expresses a first protein that reduces the amount of shikimatethat is available for the lignin biosynthesis pathway and a secondprotein that reduces the amount of phenylalanine that is available forthe lignin biosynthesis pathway. In some embodiments, the two or moreproteins potentiate or activate the same pathway; for example, in someembodiments the plant expresses a first transcription factor and asecond transcription factor that function cooperatively to induce theflavonoid biosynthesis pathway.

Proteins that Produce a Competitive Inhibitor of HCT

In some embodiments, a plant having reduced lignin content is engineeredby expressing or overexpressing a competitive inhibitor of a ligninbiosynthesis pathway enzyme (e.g., a molecule that competes withp-coumaroyl-CoA and/or shikimate as a substrate for hydroxycinnamoyl-CoAshikimate/quinate hydroxycinnamoyltransferase (HCT)). In someembodiments, the method comprises:

-   -   introducing into the plant an expression cassette comprising a        polynucleotide that encodes a protein that produces a        competitive inhibitor of hydroxycinnamoyl-CoA shikimate/quinate        hydroxycinnamoyltransferase (HCT) in the plant, wherein the        polynucleotide is operably linked to a heterologous promoter;        and    -   culturing the plant under conditions in which the protein that        produces a competitive inhibitor of HCT is expressed.

In some embodiments, the protein directly or indirectly produces one ormore of the competitive inhibitors protocatechuate, gentisate, catechol,2,3-dihydroxybenzoate, 3,6-dihydroxybenzoate, or3-hydroxy-2-aminobenzoate (e.g., by catalyzing the formation of thecompetitive inhibitor or by catalyzing the formation of a precursor tothe competitive inhibitor). Examples of pathways to produce competitiveinhibitors of HCT are shown in FIG. 27.

As a non-limiting example, in some embodiments, the competitiveinhibitor of HCT is protocatechuate. As shown in FIG. 27,protocatechuate can be produced by the enzyme dehydroshikimatedehydratase (QsuB) or by the enzyme dehydroshikimate dehydratase (DsDH).In some embodiments, the protein that produces a competitive inhibitorof HCT is a Corynebacterium glutamicum dehydroshikimate dehydratase(QsuB) having the amino acid sequence set forth in SEQ ID NO:6 or aPodospora anserina dehydroshikimate dehydratase (DsDH) having the aminoacid sequence set forth in SEQ ID NO:8. In some embodiments, the proteinis substantially identical (e.g., at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical) to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8.In some embodiments, the protein is a homolog of a Corynebacteriumglutamicum dehydroshikimate dehydratase (QsuB) having the amino acidsequence set forth in SEQ ID NO:6 or a homolog of the Podospora anserinadehydroshikimate dehydratase (DsDH) having the amino acid sequence setforth in SEQ ID NO:8. In some embodiments, a polynucleotide encoding thedehydroshikimate dehydratase comprises a polynucleotide sequence that isidentical or substantially identical (e.g., at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical) to SEQ ID NO:5 or SEQ ID NO:7.

B. Plastidial Expression of Proteins

In some embodiments, the protein that diverts a monolignol precursorfrom a lignin biosynthesis pathway as described herein is expressed inone or more specific organelles of the plant, e.g., in the plastid ofthe plant. The polynucleotide sequence encoding the protein that divertsa monolignol precursor from a lignin biosynthesis pathway (e.g., apolynucleotide encoding shikimate kinase (AroK), pentafunctional AROMpolypeptide (ARO1), dehydroshikimate dehydratase (DsDH),dehydroshikimate dehydratase (QsuB), phenylacetaldehyde synthase (PAAS),or phenylalanine aminomutase (PAM), e.g., a polynucleotide comprising asequence that is identical or substantially identical to apolynucleotide sequence of SEQ ID NO:1, 3, 5, 7, or 9, or apolynucleotide comprising a sequence that encodes a polypeptide isidentical or substantially identical to an amino acid sequence of SEQ IDNO:2, 4, 6, 8, 10, or 29) can be engineered to include a sequence thatencodes a targeting or transit signal for the organelle, e.g., atargeting or transit signal for the plastid. Targeting or transitsignals act by facilitating transport of proteins through intracellularmembranes, e.g., vacuole, vesicle, plastid, and mitochondrial membranes.

In some embodiments, the plastid targeting signal is a targeting signaldescribed in U.S. Pat. No. 5,510,471, incorporated by reference herein.In some embodiments, the plastid targeting signal is identical orsubstantially identical (e.g., at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical) to an amino acid sequence of SEQ ID NO:16. In someembodiments, the plastid targeting signal is identical or substantiallyidentical (e.g., at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical) to apolynucleotide sequence of SEQ ID NO:15. In some embodiments, theorganelle targeting signal (e.g., the plastid targeting signal) islinked in-frame with the coding sequence for the protein that diverts amonolignol precursor from a lignin biosynthesis pathway.

C. Promoters

In some embodiments, the polynucleotide encoding the protein thatdiverts a monolignol precursor from the lignin biosynthesis pathway, orthe protein that produces a competitive inhibitor of HCT, is operablylinked to a heterologous promoter. In some embodiments, the promoter isa cell- or tissue-specific promoter as described below. In someembodiments, the promoter is from a gene in the lignin biosynthesispathway (e.g., a promoter from a gene expressed in the pathway shown inFIG. 1). In some embodiments, the promoter is from a gene in the ligninbiosynthesis pathway, with the proviso that the promoter is not thenative promoter of the polynucleotide encoding the protein that divertsa monolignol precursor from the lignin biosynthesis pathway or thenative promoter of the polynucleotide encoding the protein that producesa competitive inhibitor of HCT to be expressed in the plant. In someembodiments, the promoter is a C4H, C3H, HCT, CCR1, CAD4, CAD5, FSH,PAL1, PAL2, 4CL1, or CCoAMT promoter. In some embodiments, the promoteris identical or substantially identical to a polynucleotide sequence ofany of SEQ ID NOs:18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28.

Cell- or Tissue-Specific Promoters

In some embodiments, the polynucleotide encoding the protein thatdiverts a monolignol precursor from the lignin biosynthesis pathway, orthe protein that produces a competitive inhibitor of HCT, is operablylinked to a tissue-specific or cell-specific promoter. In someembodiments, the promoter is a secondary cell wall-specific promoter ora fiber cell-specific promoter. The secondary cell wall-specificpromoter is heterologous to the polynucleotide encoding the protein thatdiverts a monolignol precursor from the lignin biosynthesis pathway,e.g., the promoter and the promoter coding sequence are derived from twodifferent species. A promoter is suitable for use as a secondary cellwall-specific promoter if the promoter is expressed strongly in thesecondary cell wall, e.g., in vessel and fiber cells of the plant, butis expressed at a much lower level or not expressed in cells without thesecondary cell wall. A promoter is suitable for use as a fibercell-specific promoter if the promoter is expressed strongly in fibercells as compared to other non-fiber cells of the plant.

In some embodiments, the promoter is an IRX5 promoter. IRX5 is a geneencoding a secondary cell wall cellulose synthase Cesa4/IRX5, (GenbankAccession No. AF458083_1). In some embodiments, the promoter isidentical or substantially identical to the pTRX5 polynucleotidesequence of SEQ ID NO:17.

Secondary cell wall-specific promoters are also described in the art.See, for example, Mitsuda et al., Plant Cell 17:2993-3006 (2005);Mitsuda et al., Plant Cell 19:270-280 (2007); and Ohtani et al., PlantJournal 67:499-512 (2011).

It will be appreciated by one of skill in the art that a promoter regioncan tolerate considerable variation without diminution of activity.Thus, in some embodiments, a promoter (e.g., a promoter from the ligninbiosynthesis pathway, a secondary cell wall-specific promoter, or afiber cell-specific promoter) is substantially identical (e.g., at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical) to a polynucleotidesequence of any of SEQ ID NOs:17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, or 28. The effectiveness of a promoter may be confirmed using areporter gene (e.g., β-glucuronidase or GUS) assay known in the art.

D. Preparation of Recombinant Expression Vectors

Once the promoter sequence and the coding sequence for the gene ofinterest (e.g., coding for a protein that diverts a monolignol precursorfrom the lignin biosynthesis pathway) are obtained, the sequences can beused to prepare an expression cassette for expressing the gene ofinterest in a transgenic plant. Typically, plant transformation vectorsinclude one or more cloned plant coding sequences (genomic or cDNA)under the transcriptional control of 5′ and 3′ regulatory sequences anda dominant selectable marker. Such plant transformation vectors may alsocontain a promoter (e.g., a secondary cell wall-specific promoter orfiber cell-specific promoter as described herein), a transcriptioninitiation start site, an RNA processing signal (such as intron splicesites), a transcription termination site, and/or a polyadenylationsignal.

The plant expression vectors may include RNA processing signals that maybe positioned within, upstream, or downstream of the coding sequence. Inaddition, the expression vectors may include regulatory sequences fromthe 3′-untranslated region of plant genes, e.g., a 3′ terminator regionto increase mRNA stability of the mRNA, such as the PI-II terminatorregion of potato or the octopine or nopaline synthase 3′ terminatorregions.

Plant expression vectors routinely also include dominant selectablemarker genes to allow for the ready selection of transformants. Suchgenes include those encoding antibiotic resistance genes (e.g.,resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin orspectinomycin), herbicide resistance genes (e.g., phosphinothricinacetyltransferase), and genes encoding positive selection enzymes (e.g.mannose isomerase).

Once an expression cassette comprising a polynucleotide encoding theprotein that diverts a monolignol precursor from the lignin biosynthesispathway and operably linked to a promoter as described herein has beenconstructed, standard techniques may be used to introduce thepolynucleotide into a plant in order to modify gene expression. See,e.g., protocols described in Ammirato et al. (1984) Handbook of PlantCell Culture—Crop Species. Macmillan Publ. Co. Shimamoto et al. (1989)Nature 338:274-276; Fromm et al. (1990) Bio/Technology 8:833-839; andVasil et al. (1990) Bio/Technology 8:429-434.

Transformation and regeneration of plants are known in the art, and theselection of the most appropriate transformation technique will bedetermined by the practitioner. Suitable methods may include, but arenot limited to: electroporation of plant protoplasts; liposome-mediatedtransformation; polyethylene glycol (PEG) mediated transformation;transformation using viruses; micro-injection of plant cells;micro-projectile bombardment of plant cells; vacuum infiltration; andAgrobacterium tumeficiens mediated transformation. Transformation meansintroducing a nucleotide sequence in a plant in a manner to cause stableor transient expression of the sequence. Examples of these methods invarious plants include: U.S. Pat. Nos. 5,571,706; 5,677,175; 5,510,471;5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,780,708;5,538,880; 5,773,269; 5,736,369 and 5,610,042.

Following transformation, plants can be selected using a dominantselectable marker incorporated into the transformation vector.Typically, such a marker will confer antibiotic or herbicide resistanceon the transformed plants or the ability to grow on a specificsubstrate, and selection of transformants can be accomplished byexposing the plants to appropriate concentrations of the antibiotic,herbicide, or substrate.

The polynucleotides coding for a protein that diverts a monolignolprecursor from the lignin biosynthesis pathway, as well as thepolynucleotides comprising promoter sequences for secondary cellwall-specific promoters or fiber cell-specific promoters, can beobtained according to any method known in the art. Such methods caninvolve amplification reactions such as PCR and otherhybridization-based reactions or can be directly synthesized.

E. Plants in which Lignin Content can be Reduced

An expression cassette comprising a polynucleotide encoding the proteinthat diverts a monolignol precursor from the lignin biosynthesis pathwayand operably linked to a promoter, or comprising a polynucleotideencoding the protein that produces a competitive inhibitor of HCT andoperably linked to a promoter, as described herein, can be expressed invarious kinds of plants. The plant may be a monocotyledonous plant or adicotyledonous plant. In some embodiments of the invention, the plant isa green field plant. In some embodiments, the plant is a gymnosperm orconifer.

In some embodiments, the plant is a plant that is suitable forgenerating biomass. Examples of suitable plants include, but are notlimited to, Arabidopsis, poplar, eucalyptus, rice, corn, switchgrass,sorghum, millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy,barley, turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow,Jatropha, and Brachypodium.

In some embodiments, the plant into which the expression cassette isintroduced is the same species of plant as the promoter and/or as thepolynucleotide encoding the protein that diverts a monolignol precursorfrom the lignin biosynthesis pathway or encoding the protein thatproduces a competitive inhibitor of HCT (e.g., a polynucleotide encodingthe protein that diverts a monolignol precursor from the ligninbiosynthesis pathway and a secondary cell wall-specific or fibercell-specific promoter from Arabidopsis is expressed in an Arabidopsisplant). In some embodiments, the plant into which the expressioncassette is introduced is a different species of plant than the promoterand/or than the polynucleotide encoding the protein that diverts amonolignol precursor from the lignin biosynthesis pathway (e.g., apolynucleotide encoding the protein that diverts a monolignol precursorfrom the lignin biosynthesis pathway and/or a secondary cellwall-specific or fiber cell-specific promoter from Arabidopsis isexpressed in a poplar plant). See, e.g., McCarthy et al., Plant CellPhysiol. 51:1084-90 (2010); and Zhong et al., Plant Physiol. 152:1044-55(2010).

F. Screening for Plants Having Reduced Lignin Content

After transformed plants are selected, the plants or parts of the plantscan be evaluated to determine whether expression of the protein thatdiverts a monolignol precursor from the lignin biosynthesis pathway, orexpression of the protein that produces a competitive inhibitor of HCT,e.g., under the control of a secondary cell wall-specific promoter or afiber cell-specific promoter, can be detected, e.g., by evaluating thelevel of RNA or protein, by measuring enzymatic activity of the protein,and/or by evaluating the size, molecular weight, content, or degree ofbranching in the lignin molecules found in the plants. These analysescan be performed using any number of methods known in the art.

In some embodiments, plants are screened by evaluating the level of RNAor protein. Methods of measuring RNA expression are known in the art andinclude, for example, PCR, northern analysis, reverse-transcriptasepolymerase chain reaction (RT-PCR), and microarrays. Methods ofmeasuring protein levels are also known in the art and include, forexample, mass spectroscopy or antibody-based techniques such as ELISA,Western blotting, flow cytometry, immunofluorescence, andimmunohistochemistry.

In some embodiments, plants are screened by assessing for activity ofthe protein being expressed, and also by evaluating lignin size andcomposition. Enzymatic assays for the proteins described herein (e.g.,shikimate kinase (AroK), pentafunctional AROM polypeptide (ARO1),dehydroshikimate dehydratase (DsDH), dehydroshikimate dehydratase(QsuB), phenylacetaldehyde synthase (PAAS), phenylalanine aminomutase(PAM), p-coumarate/cinnamate carboxylmethltransferase (CCMT1), ferulicacid decarboxylase (FDC1), phenylacrylic acid decarboxylase (PDC1),2-oxoglutarate-dependent dioxygenase (C2′H), chalcone synthase (CHS),stilbene synthase (SPS), cucuminoid synthase (CUS), or benzalacetone(BAS)) are well known in the art. Lignin molecules can be assessed, forexample, by nuclear magnetic resonance (NMR), spectrophotometry,microscopy, klason lignin assays, thioacidolysis, acetyl-bromide reagentor by histochemical staining (e.g., with phloroglucinol).

As a non-limiting example, any of several methods known in the art canbe used for quantification and/or composition analysis of lignin in aplant or plant part as described herein. Lignin content can bedetermined from extract free cell wall residues using acetyl bromide orKlason methods. See, e.g., Eudes et al., Plant Biotech. J. 10:609-620(2012); Yang et al., Plant Biotech. J. (2013) (in press); and Dence etal. (eds) Lignin determination. Berlin: Springer Verlag (1992); each ofwhich is incorporated by reference herein. Extract free cell wallresidues correspond to raw biomass, which has been extensively washed toremove the ethanol soluble component. Eudes et al., Plant Biotech. J.10:609-620 (2012); Yang et al., Plant Biotech. J. (2013) (in press);Sluiter et al., Determination of structural carbohydrates and lignin inbiomass. In: Laboratory Analytical Procedure. National Renewable EnergyLaboratory, Golden, Colo., USA; and Kim et al., Bio. Res. 1:56-66(2008). Lignin composition analysis and G/S lignin subunit determinationcan be performed using any of various techniques known in the art suchas 2D 13C-H1 HSQC NMR spectroscopy (Kim and Ralph, Org. Biomol. Chem.8:576-591 (2010); Kim et al., Bio. Res. 1:56-66 (2008)); thioacidolysismethod (Lapierre et al., Plant Physiol. 119:153-164 (1999); Lapierre etal., Res. Chem. Intermed. 21:397-412 (1995); Eudes et al., PlantBiotech. J. 10:609-620 (2012)); derivatization followed by reductivecleavage method (DFRC method; Lu and Ralph, J. Agr. Food Chem 46:547-552(1998) and Lu and Ralph, J. Agr. Food Chem 45:2590-2592 (1997)) andpyrolysis-gas chromatograph method (Py-GC method; Sonoda et al., Anal.Chem. 73:5429-5435 (2001)) directly from extract free cell wall residuesor from cellulolytic enzyme lignin (CEL lignin). CEL lignin derives fromcell wall residues, which were hydrolyzed with crude cellulases todeplete the polysaccharide fraction and enrich the lignin one (Eudes etal., Plant Biotech. J. 10:609-620 (2012)).

IV. Methods of Using Plants Having Reduced Lignin Content

Plants, parts of plants, or plant biomass material from plants havingreduced lignification due to the expression of a protein that diverts amonolignol precursor from the lignin biosynthesis pathway or due to theexpression of a protein that produces a competitive inhibitor of HCT,e.g., under the control of a secondary cell wall-specific promoter or afiber cell-specific promoter, can be used for a variety of methods. Insome embodiments, the plants, parts of plants, or plant biomass materialgenerate less recalcitrant biomass for use in a conversion reaction ascompared to wild-type plants. In some embodiments, the plants, parts ofplants, or plant biomass material are used in a saccharificationreaction, e.g., enzymatic saccharification, to generate soluble sugarsat an increased level of efficiency as compared to wild-type plants. Insome embodiments, the plants, parts of plants, or plant biomass materialare used to increase biomass yield or simplify downstream processing forwood industries (such as paper, pulping, and construction) as comparedto wild-type plants. In some embodiments, the plants, parts of plants,or plant biomass material are used to increase the quality of wood forconstruction purposes. In some embodiments the plants, parts of plants,or plant biomass material can be used in a combustion reaction,gasification, pyrolysis, or polysaccharide hydrolysis (enzymatic orchemical). In some embodiments, the plants, parts of plants, or plantbiomass material are used as feed for animals (e.g., ruminants).

Methods of conversion, for example biomass gasification, are known inthe art. Briefly, in gasification plants or plant biomass material(e.g., leaves and stems) are ground into small particles and enter thegasifier along with a controlled amount of air or oxygen and steam. Theheat and pressure of the reaction break apart the chemical bonds of thebiomass, forming syngas, which is subsequently cleaned to removeimpurities such as sulfur, mercury, particulates, and trace materials.Syngas can then be converted to products such as ethanol or otherbiofuels.

Methods of enzymatic saccharification are also known in the art.Briefly, plants or plant biomass material (e.g., leaves and stems) areoptionally pre-treated with hot water, dilute alkaline, AFEX (AmmoniaFiber Explosion), ionic liquid or dilute acid, followed by enzymaticsaccharification using a mixture of cell wall hydrolytic enzymes (suchas hemicellulases, cellulases and beta-glucosidases) in buffer andincubation of the plants or plant biomass material with the enzymaticmixture. Following incubation, the yield of the saccharificationreaction can be readily determined by measuring the amount of reducingsugar released, using a standard method for sugar detection, e.g. thedinitrosalicylic acid method well known to those skilled in the art.Plants engineered in accordance with the invention provide a highersaccharificaton efficiency as compared to wild-type plants, while theplants' growth, development, or disease resistance is not negativelyimpacted.

EXAMPLES

The following examples are provided to illustrate, but not limited theclaimed invention.

Example 1: Strategies for Diverting a Monolignol Precursor from theLignin Biosynthesis Pathway

The engineered plants of the present invention express one or more genesencoding a protein that diverts a precursor component from the ligninbiosynthesis pathway (FIG. 1) to a competitive pathway. This diversionreduces the amount of lignin that is produced and increases the amountof product produced by the competitive pathway.

FIGS. 2-11 provide exemplary strategies for diverting a precursorcomponent from the lignin biosynthesis pathway. In one strategy (FIGS. 2and 3), the monolignol precursor shikimate can be reduced or depleted.For example, the amount of cytosolic and/or plastidial shikimate that isavailable for the lignin biosynthesis pathway can be reduced or depletedby expressing a shikimate kinase such as M. tuberculosis shikimatekinase (“MtAroK”), a pentafunctional arom protein such as S. cerevisiaepentafunctional arom protein (“ScAro1”), a dehydroshikimate dehydratasesuch as C. glutamicum dehydroshikimate dehydratase (“CgQsuB”), or a P.anserina dehydroshikimate dehydratase (“PaDsDH”).

In another strategy (FIGS. 4 and 5), the monolignol precursorphenylalanine can be reduced or depleted. For example, the amount ofcytosolic and/or plastidial phenylalanine that is available for thelignin biosynthesis pathway can be reduced or depleted by expressing aphenylacetaldehyde such as P. hybrida phenylacetaldehyde synthase(“PhPAAS”) or a phenylalanine aminomutase such as T. canadensisphenylalanine aminomutase (“TcPAM”).

In another strategy (FIGS. 6 and 7), the monolignol precursors cinnamateand/or p-coumarate are reduced or depleted. For example, the amount ofcytosolic cinnamate and/or p-coumarate that is available for the ligninbiosynthesis pathway can be reduced or depleted by expressing acinnamate/p-coumarate carboxyl methyltransferase such as O. basilicumcinnamate/p-coumarate carboxyl methyltransferase (“ObCCMT1”) or aphenylacrylic acid decarboxylase such as P. pentosaceus phenylacrylicdecarboxylase (“PDC”).

In another strategy (FIGS. 8-11), the monolignol precursorscoumaroyl-CoA, caffeoyl-CoA, and/or feruloyl-CoA are reduced ordepleted. For example, the amount of cytosolic coumaroyl-CoA,caffeoyl-CoA, and/or feruloyl-CoA that is available for the ligninbiosynthesis pathway can be reduced or depleted by expressing a2-oxoglutarate-dependent dioxygenase such as R. graveolens C2′H(2-oxoglutarate-dependent dioxygenase) (“RbC2′H”), a chalcone synthase(CHS), a stilbene synthase (SPS), a cucuminoid synthase (CUS), or abenzalacetone (BAS).

Example 2: Generation of Transgenic Lines Expressing QsuB or DsDH inPlastids

The promoter (pC4H) of the lignin C4H gene from Arabidopsis wassynthesized with flanking SmaI and AvrII restriction sites at the 3′ and5′ ends respectively (Genscript). The encoding sequence of thechloroplastic targeting signal peptide sequence (ctss; U.S. Pat. No.5,510,471) was codon optimized and synthesized (Genscript), thenamplified by PCR and inserted into the AvrII restriction site located atthe 5′ end of pC4H using In-Fusion cloning (Clontech). The pC4Hctss DNAfusion was then used to replace the IRX5 promoter from pTKan-pIRX5(Eudes et al. Plant Biotechnol J 10, 609-620 (2012)) using Gatewaytechnology (Invitrogen) and to generate a new pTkan-pC4Hctss-GWR3R2vector. This vector is designed to clone in-frame with the ctss sequenceany gene of interest previously cloned into a pDONR221.P3-P2 vectoraccording to the manufacturer instruction (Invitrogen).

Codon-optimized nucleotide sequences encoding for the dehydroshikimatedehydratases QsuB from Corynebacterium glutamicum (accession numberA4QB63) and DsDH from Podospora anserina (accession number CAD60599)were synthesized for expression in Arabidopsis (Genescript), cloned inpDONR221.P3-P2 gateway vector according the manufacturer instruction(Invitrogen), and transferred into pTkan-pC4Hctss-GWR3R2 by LR clonasereaction (Invitrogen) to generate the pTKan-pC4Hctss-QsuB andpTKan-pC4Hctss-DsDH binary vectors respectively. The in-frame fusions ofcttss with QsuB and DsDH encoding sequences were verified by sequencing.

Both constructs were introduced independently into WT Arabidopsis plants(ecotype Col0) via Agrobacterium tumefaciens-mediated transformation(Bechtold and Pelletier, Methods Mol Biol 82:259-266 (1998)) and severalindependent S-QsuB and S-DsDH lines harboring ctss::QsuB and ctss::DsDHgene fusions respectively were generated.

Results

Nine independent lines resistant to kanamycin and therefore harboringthe pTKan-pC4Hctss-QsuB construct (S-QsuB lines) were selected andanalyzed at the T2 generation. These lines express the dehydroshikimatedehydratase QsuB protein from Corynebacterium glutamicum fused to aplastid targeting signal peptide to address the QsuB protein in theirplastids. At the rosette stage (3-week-old), S-QsuB lines werephenotypically indistinguishable from wild-type (WT) plants (FIG. 11).The biomass from dried senesced stems collected from S-QsuB lines and WTplants was used to perform saccharification analysis. As shown on FIG.12, the amount of reducing sugars released from the biomass of all theS-QsuB lines was higher compared to the amount released from WT plants.In particular, using similar amount of cellulolytic enzyme, the S-QsuBlines #1, 4, and 9 showed improved saccharification efficiencies of upto 3.0 fold compared to WT plants (FIG. 12). Moreover, the amount ofreducing sugars released from the biomass of S-QsuB lines (#1, #4, #9)and WT plants using different loadings of cellulolytic enzyme cocktailwas investigated. As shown on FIG. 13, the saccharification efficiencywas on average 75% higher for the three S-QsuB lines although 10 timesless enzyme was used compared to WT biomass. This result shows that muchless cellulolytic enzyme is required to release similar amount of sugarsfrom the biomass of S-QsuB lines compared to that of WT plants.

Alternatively, five independent lines resistant to kanamycin andtherefore harboring the pTKan-pC4Hctss-DsDH construct (S-DsDH lines)were selected and analyzed at the T2 generation. These lines express thedehydroshikimate dehydratase DsDH protein from Podospora anserine fusedto a plastid targeting signal peptide to address the QsuB protein intheir plastids. The biomass from dried senesced stems collected fromS-DsDH lines and WT plants was used to perform saccharificationanalysis. As shown on FIG. 14, using identical amount of cellulolyticenzyme, the amount of reducing sugars released over time from thebiomass of all the S-DsDH lines was higher compared to the amountreleased from WT plants, representing an improvement of up to 1.4 foldafter 72 h of hydrolysis. Similarly to the S-QsuB lines, this resultindicates that the biomass of S-DsDH lines is less recalcitrant topolysaccharide enzymatic digestion compared to WT plants.

Example 3: Expression of a Bacterial 3-Dehydroshikimate DehydrataseReduces Lignin Content and Improves Biomass Saccharification EfficiencyAbstract

Lignin confers recalcitrance to plant biomass used as feedstocks inagro-processing industries or as a source of renewable sugars for theproduction of bioproducts. The metabolic steps for the synthesis oflignin building blocks belong to the shikimate and phenylpropanoidpathways. Genetic engineering efforts to reduce lignin content typicallyemploy gene-knockout or gene-silencing techniques to constitutivelyrepress one of these metabolic pathways. In this study, we report thatexpression of a 3-dehydroshikimate dehydratase (QsuB fromCorynebacterium glutamicum) reduces lignin deposition in Arabidopsiscell walls. QsuB was targeted to the plastids to convert3-dehydroshikimate—an intermediate of the shikimate pathway—intoprotocatechuate. Compared to wild-type plants, lines expressing QsuBcontain higher amounts of protocatechuate, cinnamate, p-coumarate,p-coumaraldehyde, and coumaryl alcohol. 2D-NMR spectroscopy,thioacidolysis, and pyrolysis-gas chromatography/mass spectrometry(pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reductionof guaiacyl units in the lignin of QsuB lines, while size-exclusionchromatography indicates a lower degree of lignin polymerization. Ourdata show that the expression of QsuB primarily affects one of the keyenzymatic steps within the lignin biosynthetic pathway. Finally, biomassfrom these lines exhibits more than a twofold improvement insaccharification efficiency. We conclude that the expression of QsuB inplants, in combination with specific promoters, is a promisinggain-of-function strategy for spatio-temporal reduction of lignin inplant biomass.

Significance

Lignin is a complex aromatic polymer found in plant cells walls that islargely responsible for the strength and toughness of wood. Theseproperties also confer “recalcitrance” to biomass, so materials high inlignin content are more difficult to break down in processes such asproduction of biofuels. Efforts to reduce lignin content throughaltering plant gene expression often result in reduced biomass yield andcompromise plant fitness. In this study, we present an effectivealternative strategy: reducing lignin content and biomass recalcitrancethrough expression of a bacterial 3-dehydroshikimate dehydratase inplants. We demonstrate that this strategy achieved dramatic changes inthe lignin composition and structure in transgenic plants, as well asimproved conversion of biomass into fermentable sugars.

Introduction

Plant cells walls are the primary source of terrestrial biomass andmainly consist of cellulosic and hemicellulosic polysaccharidesimpregnated with lignins. Lignins are polymers of p-hydroxycinnamylalcohols (i.e., monolignols), which are synthesized inside the cells,exported to the cell wall, and ultimately undergo oxidativepolymerization via laccase and peroxidase activities. The mainmonolignols—p-coumaryl, coniferyl, and sinapyl alcohols—give rise to thep-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin units,respectively (1). Lignification generally confers mechanical strengthand hydrophobicity in tissues that develop secondary cell walls, such assclerenchyma (i.e., fibers) and xylem vessels. In addition to itsessential role for upright growth, lignin also serves as a physicalbarrier against pathogens that degrade cell walls (2).

Lignocellulosic biomass is used for pulp and paper manufacture, ruminantlivestock feeding, and more recently has been considered an importantsource of simple sugars for fermentative production of intermediate orspecialty chemicals and biofuels (3). It is well-documented that ligninin plant biomass negatively affects pulp yield, forage digestibility,and polysaccharide saccharification (4-6). This has prompted majorinterest in developing a better understanding of lignin biosynthesis toreduce biomass recalcitrance by modifying lignin content and/orcomposition.

The shikimate pathway, which is located in plastids in plants, providesa carbon skeleton for the synthesis of phenylalanine, the precursor ofthe cytosolic phenylpropanoid pathway responsible for the biosynthesisof monolignols (FIG. 20). All the metabolic steps and correspondingenzymes for both pathways are known and well-conserved across landplants (7-10). Classic approaches to lignin reduction have relied ongenetic modifications, such as transcript reduction and allelicvariation of specific genes from the phenylpropanoid pathway (11, 12).However, these strategies often result in undesired phenotypes—includingdwarfism, sterility, and increased susceptibly to environmentalstresses—due to loss of cell-wall integrity, depletion of otherphenylpropanoid-related metabolites, accumulation of pathwayintermediates, or the constitutive activation of defense responses (13,14). Such negative effects are unfortunately difficult to avoid becauseof the non-tissue specificity of the strategies employed: allelicvariations are transmitted to every cell of the plant during celldivisions, and small interfering RNAs generated for gene silencinggenerally move from cell-to-cell and over long distance in vegetativetissues (15).

Alternatively, there are novel and promising gain-of-function strategiesthat involve expression of specific proteins to reduce the production ofthe three main monolignols or change their ratios. Using specificpromoters with restricted expression patterns, these strategies wouldenable the alteration of lignin at later developmental stages or, forexample, only in certain tissues such as fibers—without compromising thefunctionality of conductive vessels for the transport of water (14).Examples of such expressed proteins are transcription factors that actas negative regulators of lignin biosynthesis (16-19); enzymes that useintermediates of the lignin pathway for the synthesis of derivedmetabolites (20-22); engineered enzymes that modify monolignols intotheir non-oxidizable forms (23); or proteins that mediate thepost-transcriptional degradation of enzymes from the lignin biosyntheticpathway (24).

In this study, we report for the first time on the expression of abacterial 3-dehydroshikimate dehydratase in Arabidopsis (25). Weselected QsuB from C. glutamicum and targeted it to the plastids toconvert the shikimate precursor 3-dehydroshikimate into protocatechuate,with the aim of reducing lignin content and modifying its compositionand structure in the biomass of transgenic lines. Metabolomic analysisof plants expressing QsuB revealed higher amounts of cinnamate,p-coumarate, and of the two direct precursors of H-lignin units:p-coumaraldehyde and p-coumaryl alcohol. Conversely, the directprecursors of G and S units—coniferaldehyde, coniferyl alcohol,sinapaldehyde, and sinapyl alcohol—were reduced. Lignin content wasseverely reduced in these transgenic lines and exhibited an enrichmentof H units at the expense of G units and a lower polymerization degree.Compared to those of wild-type plants, cell walls from lines expressingQsuB released significantly higher amounts of simple sugars aftercellulase treatment and required less enzyme for saccharification.Collectively, these results support the hypothesis that expression of aplastidic QsuB affects one of the enzymatic steps within the ligninbiosynthetic pathway.

Results Targeted Expression of QsuB in Arabidopsis

A sequence encoding QsuB was cloned downstream of the sequence encodingfor a plastid-targeting signal peptide (SCHL) for expression inplastids. Using transient expression in tobacco, we first confirmed thatQsuB was correctly targeted to the plastids by analyzing its subcellularlocalization when fused at the C-terminus to a YFP marker (FIG. 21). Theschl-qsuB sequence was cloned downstream of the Arabidopsis C4H promoterfor expression in lignifying tissues of Arabidopsis. Western blotanalysis confirmed that QsuB was expressed in stems of several T2 plantshomozygous for the pC4H::schl::qsuB construct (FIG. 16). Based on themigration of molecular weight markers, QsuB was detected at around 70kDa, which corresponds to the theoretical size of its native sequenceafter cleavage of the chloroplast transit peptide (FIG. 16). Five lineswith different QsuB expression levels (C4H::qsuB-1, -3, -6, -7, and -9)were selected for biomass measurement. Although a height reduction wasobserved for these lines, only two of them (C4H::qsuB-1 and -9) showed aslight decrease of biomass yield (stem dry weight) by 18% and 21%,respectively (Table 1).

TABLE 1 Height and dry weight of the main inflorescence stem of senescedmature wild-type (WT) and pC4H::schl::qsuB (C4H::qsuB) plants. Height(cm) Dry weight (mg) Plant line Mean ± SE Mean ± SE n WT 47.3 ± 0.8  271.0 ± 11.1 24 C4H::qsuB-1 36.6 ± 1.0***  221.3 ± 11.0** 20 C4H::qsuB-338.8 ± 0.7*** 244.4 ± 13.4 20 C4H::qsuB-6 35.9 ± 0.9*** 254.1 ± 12.7 20C4H::qsuB-7 41.0 ± 0.9*** 251.3 ± 17.4 20 C4H::qsuB-9 31.8 ± 0.7*** 214.4 ± 14.2** 20 n = number of plants analyzed. Asterisks indicatesignificant differences from the wild-type using the unpaired Student'st-test (*P < 0.05; **P < 0.005; ***P < 0.001).Metabolite Analysis of C4H::qsuB Lines

Methanol soluble metabolites from stems of the C4H::qsuB-1 andC4H::qsuB-9 lines were extracted for analysis (Table 2, FIG. 22).Compared to wild-type plants, protocatechuate content was increased 53-and 485-fold in those two transgenic lines, respectively. However,except for tyrosine in line C4H::qsuB-9, no significant reduction wasobserved for the content of several metabolites derived from theshikimate pathway in plastids such as salicylate and aromatic aminoacids. Instead, salicylate was slightly increased, 1.3-1.4-fold, in bothlines and phenylalanine was 1.6-fold higher in line C4H::qsuB-1.Interestingly, several metabolites from the phenylpropanoid pathway wereincreased in the transgenic lines. Cinnamate and p-coumaraldehyde weredetected only in transgenic lines; while p-coumarate and p-coumarylalcohol contents were increased, compared to those of wild type,14-18-fold and 3.5-30-fold, respectively. Kaempferol and quercetin, twoflavonols derived from p-coumaroyl-CoA, were also found in higheramounts in both C4H::qsuB lines. The direct precursors of G- andS-lignin units were negatively altered; coniferaldehyde was reduced ˜40%in both transgenic lines, while coniferyl alcohol, sinapaldehyde, andsinapyl alcohol were decreased twofold in C4H::qsuB-9 (Table 2).

Cell wall-bound metabolites released from cell wall residues by mildalkaline hydrolysis were also analyzed (Table 3). Protocatechuate wasfound in cell walls of the C4H::qsuB lines but not in those fromwild-type plants. The content of p-coumarate was significantly increasedin line C4H::qsuB-1, whereas ferulate was reduced in both transgeniclines.

TABLE 2 Quantitative analysis of methanol-soluble metabolites in stemsfrom 6-wk-old wild-type (WT) and pC4H::schl::qsuB (C4H::qsuB) plants.Mean ± SE Metabolites WT C4H::qsuB-1 C4H::qsuB-9 Protocatechuate^(α)2.04 ± 0.4   108.0 ± 24.8****   991.9 ± 60.7**** Tryptophan^(α)  3.7 ±0.5 3.4 ± 0.2 3.4 ± 0.2 Phenylalanine^(α)  2.9 ± 0.2   4.7 ± 0.2*** 3.3± 0.2 Tyrosine^(α)  5.0 ± 1.1 4.2 ± 0.6  2.7 ± 0.2* Sinapyl alcohol^(α) 4.1 ± 0.3  5.7 ± 0.4**   1.9 ± 0.4*** Quercetin^(α) 16.1 ± 3.6 12.8 ±0.6  24.6 ± 1.8* Kaempferol^(α) 159.4 ± 31.6 239.8 ± 9.7** 260.2 ± 8.8**p-Coumarate^(β)  6.8 ± 1.2  123.1 ± 9.9****   93.7 ± 12.8**** p-Coumarylalcohol^(β)  7.6 ± 1.9  26.8 ± 4.8**   229.6 ± 32.8**** Coniferylaldehyde^(β) 28.6 ± 1.8  18.1 ± 2.3**  16.6 ± 1.8*** Coniferylalcohol^(β) 828.5 ± 99.2 671.0 ± 63.2   457.0 ± 62.2** Sinapylaldehyde^(β) 59.2 ± 3.9 68.1 ± 8.7   36.4 ± 3.1*** Salicylate^(β) 655.3± 30.7  854.4 ± 63.1**  905.7 ± 111.5* Cinnamate^(β) nd^(φ) 977.2 ±389.1 144.3 ± 50.5  ^(α)(μg/g fresh weight) ^(β)(μg/g fresh weight)^(φ)Using a detection limit of 34 ng/g fresh weight Values are means offour biological replicates (n = 4). nd, not detected. Asterisks indicatesignificant differences from the wild type using the unpaired Student'st-test (*P < 0.1; **P < 0.05; ***P < 0.005; ****P < 0.001).

TABLE 3 Quantitative analysis of cell wall-bound aromatics in stems fromextractive-free senesced mature wild- type (WT) and pC4H::schl::qsuB(C4H::qsuB) plants. Mean ± SE (μg/g dry weight) Metabolite WTC4H::qsuB-1 C4H::qsuB-9 Protocatechuate nd 6.3 ± 0.4 6.7 ± 1.4p-Coumarate 15.8 ± 3.0 32.4 ± 2.5* 20.4 ± 1.0  Ferulate 18.1 ± 0.7  7.8± 0.5**  5.3 ± 0.1** Values are means of four biological replicates (n =3). nd, not detected. Asterisks indicate significant differences fromthe wild type using the unpaired Student's t-test (*P < 0.05; **P <0.005; ***P < 0.001).Compositional Analysis of Cell Wall from C4H::qsuB Lines

Using the Klason method, the lignin content measured in the stem oflines C4H::qsuB-1 and C4H::qsuB-9 was reduced 50% and 64%, respectively,compared to that of wild type (Table 4). Analysis of the cell-wallmonosaccharide composition showed higher amounts of glucose (+4-10%),xylose (+13-19%), and other less abundant sugars in the transgeniclines, resulting in 8% increase in total cell-wall sugars for theC4H::qsuB-1 line and an 11% increase for C4H::qsuB-9 line (Table 4).

TABLE 4 Chemical composition of senesced mature stems from wild- type(WT) and pC4H::schl::qsuB (C4H::qsuB) plants. Mean ± SE (mg/g cell wall)Component WT C4H::qsuB-1 C4H::qsuB-9 Glucose 376.7 ± 5.0 391.6 ± 2.9* 416.0 ± 0.9** Xylose 173.0 ± 2.0 199.5 ± 2.2** 212.9 ± 0.2**Galacturonic acid  60.8 ± 2.0 70.8 ± 0.5* 63.1 ±0.8  Galactose  20.5 ±0.5 23.3 ±0.1*  20.2 ± 0.3  Arabinose  17.1 ± 0.4 19.4 ± 0.1* 16.8 ±0.3  Rhamnose  12.1 ± 0.3  14.1 ± 0.2** 13.0 ± 0.2  Fucose  1.8 ± 0.12.3 ± 0.1 2.0 ± 0.1 Glucuronic acid  7.1 ± 0.1 7.3 ± 0.1  8.2 ± 0.2*Total sugars 669.1 ± 6.8 728.4 ± 5.1** 752.3 ± 2.8** Klason lignin 191.5± 9.5  96.2 ± 8.0**  68.4 ± 5.8** Acid soluble lignin  4.5 ± 0.4 5.0 ±0.7 4.7 ± 0.9 Values are means ± SE of triplicate analyses (n = 3).Asterisks indicate significant differences from the wild type using theunpaired Student's t-test (*P < 0.05; **P < 0.005).Lignin Monomeric Composition and Structure in C4H::qsuB Lines

Determination of the lignin monomer composition, using thioacidolysis,indicated an increase in the relative amount of H units in transgeniclines. H units represented 12.7% and 27.9% of the total lignin monomersin lines C4H::qsuB-1 and C4H::qsuB-9, which corresponds to 21- and46-fold increases compared to that of wild type, respectively (Table 5).The relative amount of G units in transgenics (˜45%) was also reducedcompared to wild type (˜64%), whereas S units were higher in C4H::qsuB-1and lower in C4H::qsuB-9 (Table 5).

NMR (2D ¹³C-¹H-correlated, HSQC) spectra of cell-wall material fromC4H::qsuB-1 and C4H::qsuB-9 lines were also obtained for determinationof lignin composition and structure. Analysis of the aromatic region ofthe spectra confirmed the higher relative amount of H units in bothC4H::qsuB lines (29% and 64.4% respectively) compared to that in wildtype (3.6%), as well as a reduction of G units (FIG. 17). Moreover,analysis of the aliphatic region of the spectra indicated a strongreduction of phenylcoumaran (β-5) and resinol (β-β) linkages in thelignin of the transgenic lines (FIG. 23).

Finally, cell-wall material from stems of wild-type and C4H::qsuB lineswere analyzed by pyro-GC/MS. For each line, identification and relativequantification of the pyrolysis products derived from H, G, or S unitsallowed determination of H/G/S ratios (FIG. 28). Compared to wild type,H units were increased 3.5- and 10-fold, and G units were reduced 1.4-and 2.2-fold, in lines C4H::qsuB-1 and C4H::qsuB-9, respectively.

TABLE 5 Main H, G, and S lignin-derived monomers obtained bythioacidolysis of extractive-free senesced mature stems from wild-type(WT) and pC4H::schl::qsuB (C4H::qsuB) plants. WT C4H::qsuB-1 C4H::qsuB-9Total yield (μmol/g CWR) 263.5 (22.7) 116.3 (11.8)* 73.5 (2.1)** Totalyield (μmol/g KL) 1372.5 (118.5) 1211.8 (122.6) 1081.2 (30.7)* % H 0.6(0.03) 12.7 (0.78)** 27. 9 (0.38)** % G 63.7 (0.46) 46.5 (1.94)* 44.9(0.40)* % S 35.7 (0.43) 40.8 (1.16)* 27.2 (0.02)* Values in parenthesesare the SE from duplicate analyses. Asterisks indicate significantdifferences from the wild type using the unpaired Student's t-test (*P <0.05; **P < 0.01).Lignins from C4H::qsuB Lines have a Lower Polymerization Degree

Lignin fractions were isolated from wild-type and C4H::qsuB lines foranalysis of their polydispersity using size-exclusion chromatography(SEC). Elution profiles acquired by monitoring UV-F fluorescence of thedissolved lignin revealed differences between wild-type and transgeniclines (FIG. 18). The total area of the three mass peaks, correspondingto the largest lignin fragments detected between 7.8 min and 12.5 min,was significantly reduced in C4H::qsuB lines compared to wild type.Similarly, intermediate molecular mass material, which elutes in afourth peak between 12.5 min and 18 min, was also less abundant inC4H::qsuB lines. Conversely, the area corresponding to the smallestlignin fragments, detected between 18 min and 23.5 min, was increased inthe transgenic lines. These results demonstrate a reduction in thedegree of polymerization of lignins purified from plants expressing QsuBcompared to that of wild type.

Biomass from C4H::qsuB Lines Shows Improved Saccharification

Saccharification assays on stem material were conducted to evaluate thecell-wall recalcitrance of the C4H::qsuB lines. As shown in FIG. 19A,higher amounts of sugars were released after 72 hr enzymatic hydrolysisof biomass from the C4H::qsuB lines (−1, −3, −6, −7 and −9) compared tothose of wild type in all pretreatments tested. Saccharificationimprovements ranged between 79-130% after hot water; 63-104% afterdilute alkali; and 26-40% after dilute acid pretreatments (FIG. 19A).Moreover, similar saccharification experiments using hot waterpretreated biomass, at 5× lower cellulase loadings, revealed thatbiomass from all C4H::qsuB lines releases more sugar than that of wildtype hydrolyzed with a typical enzyme loading (FIG. 19B). Takentogether, these data demonstrate that cellulose from the C4H::qsuB linesis less recalcitrant to cellulase digestion and requires a lower amountof enzyme to be converted into high yields of fermentable sugars.

Discussion

Gain-of-function strategies have several advantages for the manipulationof metabolic pathways. For example, they can be used to bioengineerlignin deposition in plants via better spatio-temporal control ofmonolignol production in lignifying cells, and to adjust lignincomposition and its biophysical properties (26). Therefore,identification of proteins in which in planta-expression results inmodifications of lignin content or composition is of particular interestand presents novel opportunities. In this work, we demonstrate thatexpression of the 3-dehydroshikimate dehydratase QsuB in plastids leadsto drastic reduction and compositional changes of lignin in Arabidopsis(Table 4). As a result, biomass from these transgenic plants exhibitsmuch higher saccharification efficiency after pretreatment (FIG. 19A),which is a highly desired trait for several agro-industries and thebioenergy sector. Moreover, the efficiency of this approach to decreaselignin content in plant biomass allows a reduction of hydrolytic enzymeloadings by at least five-fold, while retaining greater saccharificationpotential than control plants hydrolyzed at standard enzyme loading(FIG. 19B). Consequently, the transfer of this technology to energycrops should have a great impact on the cost-effectiveness of cellulosicbiofuels production, since enzyme cost is the major barrier in thisprocess (27).

In this study, as a proof of concept, we used the promoter of the AtC4Hgene to ensure strong QsuB expression in all lignifying tissues of theplant. This resulted in a slight decrease of plant height for all thelines; but no significant reductions in biomass yield except for that oftwo transgenic lines, which expressed QsuB very strongly (Table 1; FIG.16) and exhibited in some stem transverse sections (FIG. 24) evidence ofvessel collapse that could impair xylem conductivity (14). Nevertheless,our strategy offers the potential to overcome these defects by selectingmore stringent promoters (e.g., fiber-specific) that would exclude QsuBexpression from xylem-conductive elements (26, 28). Moreover,translation of our technology from model plant to crops is expected tobe straightforward: it is based solely on the expression of QsuB, doesnot require any particular genetic backgrounds, and the lignin andshikimate pathways are well-conserved among vascular plants.

A direct consequence of QsuB expression is the accumulation ofprotocatechuate in the biomass of transgenic plants (˜1% dry weight inline C4H::qsuB-9; Table 2). Considering the beneficial properties ofprotocatechuate in the bio-based polymer industry and human healthsector, such de novo production adds extra commercial value to thebiomass of plants expressing QsuB (29, 30). Much higher amounts ofprotocatechuate were recovered after acid treatment of themethanol-soluble extracts from transgenic plants (data not shown), whichsuggests its conjugation in the cytosol after export from the plastids.Interestingly, QsuB expression did not affect substantially the level ofmetabolites derived from the shikimate pathway, such as aromatic aminoacids and salicylate, suggesting that plastidic 3-dehydroshikimate isnot limiting (Table 2). On the other hand, a buildup of cinnamate andp-coumarate was observed in these lines, accompanied by an accumulationof p-coumaraldehyde and p-coumaryl alcohol pools (Table 2 and FIG. 22).

Analysis of the lignin monomeric composition using 2D NMR spectroscopy,thioacidolysis, and pyro-GC/MS unequivocally demonstrated an increase inH units in plants expressing QsuB (FIG. 17 and FIG. 28; Table 5). Thesedata could explain the reduced degree of polymerization of theselignins, which has been previously observed in various lignin mutantsthat exhibit high content of H units, incorporation of which typicallyslows or stops lignin-chain elongation (31, 32; FIG. 18). Therefore,reduced lignin-polysaccharide crosslinking within the biomass of thetransgenic lines is expected, and this could contribute to its superiorenzymatic digestibility.

A low lignin content rich in H-units corresponds to a phenotypepreviously characterized in plants down-regulated forhydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase(HCT), p-coumarate 3-hydroxylase (C3H), or caffeoyl shikimate esterase(CSE). This suggests that an alteration of these biosynthetic steps hasoccurred in the C4H::qsuB lines (10, 32, 33). A possible explanation isthat QsuB activity in plastids affects the export of shikimate from theplastids to the cytosol. This would indirectly limit the availability ofcytosolic shikimate used for the enzymatic step catalyzed by HCT. Thedistribution of shikimate between plastids and the cytosol is stillpoorly understood, and shikimate levels were below the detection limitin our stem extracts from wild-type and transgenic plants.Alternatively, because previous studies reported a substrate flexibilityof HCTs (34, 35), the large accumulation of protochatechuate could actas inhibitor of AtHCT, which couples p-coumaroyl-CoA and shikimate.Using an in vivo enzymatic assay to determine the substrate preferenceof AtHCT, we confirmed its affinity forp-coumaroyl-CoA and shikimate,but also demonstrated its capacity to accept protocatechuate and severalother substrates such as catechol, 3,6-dihydroxybenzoate,3-hydroxy-2-aminobenzoate, and 2,3-dihydroxybenzoate (FIG. 25).Therefore, we cannot exclude the possibility that the protocatechuatepool accumulated in C4H::qsuB plants exerts a competitive inhibition ofHCT and limits the synthesis of coumaroyl shikimate required for theproduction of G- and S-lignin units.

Materials and Methods Plant Material and Growth Conditions

Arabidopsis thaliana (ecotype Columbia, Col-0) seeds were germinateddirectly on soil. Growing conditions were 150 μmol/m²/s, 22° C., 60%humidity, and 10 h of light per day for the first 4-5 wk, followed by 14h of light per day until senescence. Selection of T1 and T2 transgenicplants was made on Murashige and Skoog vitamin medium (PhytoTechnologyLaboratories, Shawnee Mission, Kans.), supplemented with 1% sucrose,1.5% agar, and 50 μg/mL kanamycin.

Generation of Binary Vectors

The promoter p35S, with a single enhancer, was amplified by PCR frompRT100 with phosphorylated primers F-p35S(5′-GTCAACATGGTGGAGCACGACAC-3′; SEQ ID NO:46) and R-p35S(5′-CGAGAATCTAGATTGTCCTCTCCAAATGAAATGAACTTC-3′; SEQ ID NO:47), andcloned into a SmaI-digested dephosphorylated pTkan vector (36) togenerate a pTKan-p35S vector. Subsequently, a GW-YFP cassette wasextracted from the pX-YFP vector (37) by XhoI/SpeI digestion, andligated into a XhoISpeI-digested pTKan-p35S vector to generate thepTkan-p35S-GWR1R2-YFP vector.

A chimeric DNA construct was synthesized (GenScript, Piscatway, N.J.):it was flanked by the gateway sequences attB4r (5′-end) and attB3r(3′-end), and contained, in the following order, the tG7 terminator; therestriction sites SmaI, KpnI, HindIII and XhoI; a 2.9-Kb sequencecorresponding to the Arabidopsis C4H promoter (pC4H); and a sequenceencoding a plastid targeting signal (SCHL; 38). ThisattB4r-tG7-pC4H-schl-attB3r construct was then subcloned into theGateway pDONR221-P4rP3r entry vector by BP recombination (Lifetechnologies, Foster City, Calif., USA) to generatepENTR-L4-tG7-pC4H-schl-L3. An LR recombination reaction was performedwith pTkan-pIRX5-GW (21), pENTR-L1-pLac-lacZalpha-L4 (Life technologies,Foster City, Calif., USA), pENTR-L3-pLac-Tet-L2 (Life technologies,Foster City, Calif., USA), and pENTR-L4-tG7-pC4H::schl-L3. The obtainedconstruct was subsequently digested by SmaI to remove the pLac-lacZalphaand tG7 fragments. The pLac-Tet fragment was replaced by the gatewaycassette using BP recombination to generate the pTKan-pC4H::schl-GWR3R2vector.

Generation of a pTkan-pC4H::Schl-qsuB Plasmid and Plant Transformation

A gene sequence encoding QsuB from C. glutamicum (GenBank accessionnumber YP_001137362.1) without stop codon and flanked with the GatewayattB3 (5′-end) and attB2 (3′-end) recombination sites was synthesizedfor expression in Arabidopsis (GenScript, Piscatway, N.J.) and clonedinto the Gateway pDONR221-P3P2 entry vector by BP recombination (Lifetechnologies, Foster City, Calif., USA). A sequence-verified entry clonewas LR recombined with the pTKan-pC4H::schl-GWR3R2 vector to generatethe pTKan-pC4H::schl-qsuB construct, which was introduced into wild-typeArabidopsis plants (ecotype Col-0) via Agrobacterium-mediatedtransformation (39).

Western Blot Analysis

Proteins from Arabidopsis stems were extracted using a buffer containing250 mM Tris-HCl pH 8.5, 25 mM EDTA, 2 mM DTT, 5 mM β-mercaptoethanol,and 10% sucrose; and were quantified using the Bradford method (40).Proteins (15 μg) were separated by SDS-PAGE, blotted, and immunodetectedusing a universal antibody, as previously described (41).

Methanol-Soluble Metabolites Extraction

Arabidopsis stems of 6-wk-old wild-type and transgenic lines werecollected in liquid nitrogen and stored at −80° C. until furtherutilization. Prior the metabolite extraction, collected stems werepulverized in liquid nitrogen. For extraction of methanol-solublemetabolites, 700-1,000 mg of frozen stem powder was mixed with 2 ml of80% (v/v) methanol-water and mixed (1,400 rpm) for 15 min at 70° C. Thisstep was repeated four times. Pooled extracts were cleared bycentrifugation (5 min, 20,000×g, at room temperature), mixed with 4 mLof analytical grade water and filtered using Amicon Ultra centrifugalfilters (10,000 Da MW cutoff regenerated cellulose membrane; EMIDMillipore, Billerica, Mass.). Filtered extracts were lyophilized and theresulting pellets dissolved in 50% (v/v) methanol-water prior to LC-MSanalysis. An acid-hydrolysis of the samples was performed for thequantification of protocatechuate, salicylate, and flavonols; an aliquotof the filtered extracts was dried under vacuum, resuspended with 1 NHCl and incubated at 95° C. for 3 h. The mixture was subjected to threeethyl acetate partitioning steps. Ethyl acetate fractions were pooled,dried in vacuo, and resuspended in 50% (v/v) methanol-water prior toLC-MS analysis.

Cell-Wall Bound Aromatics Extraction

Senesced stems were ball-milled using a Mixer Mill MM 400 (Retsch Inc.,Newtown, Pa.) and stainless steel balls for 2 min at 30 si.Extractive-free cell-wall residues (CWR) were obtained by sequentiallywashing 60 mg of ball-milled stems with 1 mL of 96% ethanol at 95° C.twice for 30 min and mixing with 1 mL of 70% ethanol twice for 30 sec.The resulting CWR were dried in vacuo overnight at 30° C. The CWR (6 mg)were mixed with 500 μL of 2 M NaOH and shaken at 1,400 rpm for 24 h at30° C. The mixture was acidified with 100 μL of concentrated HCl, andsubjected to three ethyl acetate partitioning steps. Ethyl acetatefractions were pooled, dried in vacuo, and suspended in 50% (v/v)methanol-water prior to LC-MS analysis.

LC-MS Analysis

As previously described in Bokinsky et al. (42) and Eudes et al.(43)—aromatic amino acids, and aromatic acids and aldehydes,respectively—were analyzed using high-performance liquid chromatography(HPLC), electrospray ionization (ESI), and time-of-flight (TOF) massspectrometry (MS). Aromatic alcohols were analyzed by HPLC—atmosphericpressure chemical ionization (APCI)—TOF MS. Their separation wasconducted on an Agilent 1200 Series Rapid Resolution HPLC system(Agilent Technologies Inc., Santa Clara, Calif., USA) using a PhenomenexKinetex XB-C18 (100 mm length, 2.1 mm internal diameter, and 2.6 μmparticle size; Phenomenex, Torrance, Calif., USA). The mobile phase wascomposed of 0.1% formic acid in water (solvent A) and methanol (solventB). The elution gradient was as follows: from 5% B to 25% B for 6 min,25% B to 5% B for 1 min, and held at 5% B for a further 3 min. A flowrate of 0.5 mL/min was used throughout. The column compartment andsample tray were set to 50° C. and 4° C., respectively. The HPLC systemwas coupled to an Agilent Technologies 6210 LC/TOF mass spectrometerwith a 1:4 post-column split. Mass spectrometric detection was conductedusing APCI in the positive ion mode. MS experiments were carried out inthe full scan mode, at 0.86 spectra/second, for the detection of[M-H₂O+H]⁺ ions. Drying and nebulizing gases were set to 10 L/min and 25psi, respectively, and a drying gas temperature of 330° C. was usedthroughout. The vaporizer and corona were set to 350° C. and 4 μArespectively, and a capillary voltage of 3,500 V was also used.Fragmentor and OCT 1 RF voltages were each set to 135 V, while theskimmer voltage was set to 50 V. Data acquisition and processing wereperformed by the MassHunter software package (Agilent Technologies Inc.,Santa Clara, Calif., USA). Metabolites were quantified via 10-pointcalibration curves of authentic standard compounds for which the R²coefficients were ≥0.99. The p-coumaraldehyde content was estimated byintegrating the area of the mass peak eluting at Rt=8.6 min([M-H]=131.050238) and for which the ratio [theoretical mass/observedmass] was less than ±5 ppm (FIG. 26).

Carbohydrate and Lignin Assays

For each genotype (wild type, C4H::qsuB-1, and C4H::qsuB-9), samplesconsisted of equal mixtures of stem material from three independentcultures. Biomass was extracted sequentially by sonication (20 min) with80% ethanol (three times), acetone (one time), chloroform-methanol (1:1,v/v, one time) and acetone (one time). For determination of carbohydratecomposition, the biomass was acid-hydrolyzed as previously described(44). After CaCO₃ neutralization, monomeric sugars from the biomasshydrolyzates were separated by high-performance anion exchangechromatography with pulsed amperiometric detection using a PA20 column(Dionex, Sunnyvale, Calif., USA) and quantified as previously described(45). A calibration curve of monosaccharide standards was run forverification of response factors. The standard NREL biomass protocol wasused to measure lignin and ash (46). All carbohydrate and lignin assayswere conducted in triplicate. The thioacidolysis procedure was carriedout as described (47, 48) and the lignin-derived monomers wereidentified by GC-MS as their trimethyl-silylated derivatives.

2D ¹³C-¹H Heteronuclear Single Quantum Coherence (HSQC) NMR Spectroscopy

For each genotype (wild type, C4H::qsuB-1 and C4H::qsuB-9), samplesconsisted of equal mixtures of stem material from three independentcultures. Samples were extracted and ball milled as previously described(49, 50). The gels were formed using DMSO-d₆/pyridine-d₅ (4:1) andsonicated until homogenous in a Branson 2510 table-top cleaner (BransonUltrasonic Corporation, Danbury, Conn.). The temperature of the bath wasclosely monitored and maintained below 55° C. The homogeneous solutionswere transferred to NMR tubes. HSQC spectra were acquired at 25° C.using a Bruker Avance-600 MHz instrument equipped with a 5 mminverse-gradient ¹H/¹³C cryoprobe using a hsqcetgpsisp2.2 pulse program(ns=400, ds=16, number of increments=256, d₁=1.0 s) (53). Chemicalshifts were referenced to the central DMSO peak (δ_(C)/δ_(H) 39.5/2.5ppm). Assignment of the HSQC spectra was described elsewhere (51, 54). Asemi-quantitative analysis of the volume integrals of the HSQCcorrelation peaks was performed using Bruker's Topspin 3.1 (Windows)processing software. A Guassian apodization in F₂ (LB=−0.50, GB=0.001)and squared cosine-bell in F₁ (LB=−0.10, GB=0.001) were applied prior to2D Fourier Transformation.

Isolation of Cellulolytic Enzyme Lignin

For each genotype (wild type, C4H::qsuB-1 and C4H::qsuB-9), samplesconsisted of equal mixtures of stem material from three independentcultures. The extracted biomass was ball-milled for 3 h per 500 mg ofsample (in 10 min on/10 min off cycles) using a PM100 ball mill (Retsch,Newtown, Pa.) vibrating at 600 rpm in zirconium dioxide vessels (50 mL)containing ZrO₂ ball bearings (10×10 mm). Ball-milled walls weredigested four times over 3 d at 50° C. with the polysaccharidases CellicCTec2 and HTec2 (Novozymes, Davis, Calif.) and pectinase fromAspergillus niger (Sigma-Aldrich, St. Louis, Mo.) in sodium citratebuffer (pH 5.0). The obtained cellulolytic lignin was washed withdeionized water and lyophilized overnight.

Size Exclusion Chromatography

Lignin solutions, 1% (w/v), were prepared in analytical-grade1-methyl-2-pyrrolidinone (NMP). The polydispersity of dissolved ligninwas determined using analytical techniques involving SEC UV-F_(250/400)as previously described (53). An Agilent 1200 series binary LC system(G1312B) equipped with diode-array (G1315D) and fluorescence (G1321A)detectors was used. Separation was achieved with a Mixed-D column (5 μmparticle size, 300 mm×7.5 mm i.d., linear molecular mass range of 200 to400,000 u, Agilent Technologies Inc.) at 80° C. using a mobile phase ofNMP at a flow rate of 0.5 ml/min. Absorbance of materials eluting fromthe column was detected using UV-F fluorescence (Ex₂₅₀/Em₄₅₀). Spectralintensities were area-normalized and molecular mass estimates weredetermined after calibration of the system with polystyrene standards.

Cell Wall Pretreatments and Saccharification

Ball-milled senesced stems (10 mg) were mixed with 340 μL of water, 340μL of H₂SO₄ (1.2%, w/v), or 340 μL of NaOH (0.25%, w/v) for hot water,dilute acid, or dilute alkali pretreatments, respectively; shaken at1,400 rpm (30° C., 30 min), and autoclaved at 120° C. for 1 h. Samplespretreated with dilute acid were neutralized with 5 N NaOH (25 μL).Saccharification was initiated by adding 650 μL of 100 mM sodium citratebuffer pH 5 (for hot water- and dilute alkali-pretreated samples) or 625μL of 80 mM sodium citrate buffer pH 6.2 (for dilute acid-pretreatedsamples) containing 80 μg/mL tetracycline and 1% w/w or 0.2% w/w CellicCTec2 cellulase (Novozymes, Davis, Calif.). After 72 h of incubation at50° C. with shaking (800 rpm), samples were centrifuged (20,000×g, 3min) and 10 μL of the supernatant was collected for measurement ofreducing sugars using the 3,5-dinitrosalicylic acid assay and glucosesolutions as standards (54).

Subcellular Localization of QsuB

The schl-qsuB nucleotide sequence from the pTkan-pC4H::schl-qsuBconstruct was amplified using oligonucleotides5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCTTCGATCTCCTCCT-3′ (SEQ ID NO:48;attB1 site underlined) and5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCGTTTGGGATACCTCTCTCTAAATCT C-3′ (SEQ IDNO:49; attB2 site underlined) and cloned into the Gateway pDONR221-flentry vector (Lalonde S, et al. (2010) Front Physiol 1:24). Asequence-verified entry clone was LR recombined with thepTKan-p35S-GWR1R2-YFP vector to generate the pTKan-p35S-schl-qsuB-YFPconstruct. Infiltration of 4-wo N. benthamiana leaves was done using theAgrobacterium strain GV3101, following the method described by Sparkeset al. (Nat Protoc 1(4):2019-2025). Plants transiently expressing theSCHL-QsuB-YFP fusion protein were analyzed by confocal laser scanningmicroscopy 2 d after the infiltration. The microscopy was performedusing a Zeiss LSM 710 device (Carl Zeiss Microscopy, Jena, Germany)equipped with an argon laser (excitation at 514 nm and emissioncollected at 510 to 545 nm).

Lignin Histochemical Staining

Histochemical staining was performed as described by Pradhan-Mitra andLoqué (“Histochemical staining of Arabidopsis thaliana secondary cellwall elements,” JoVE (in press)). Basal stem transverse sections (100 μmthick) were obtained using a vibratome. Sections were incubated for 3min in phloroglucinol-HCl reagent (VWR International, Brisbane, Calif.),rinsed with water, and observed using bright field light microscopy(Leica Microsystems Inc., Buffalo Grove, Ill.).

Pyrolysis-Gas Chromatography Mass Spectrometry

Chemical composition of lignin in plant cell-wall samples were analyzedby pyrolysis-gas chromatography (GC)/mass spectrometry (MS) using apreviously described method with some modifications (Del Río J C, et al.(2012) J Agric Food Chem 60(23):5922-5935). Pyrolysis of plant cellwalls was performed with a Pyroprobe 5200 (CDS Analytical, Inc.)connected with GC/MS (Thermo Electron Corporation with Trace GC Ultraand Polaris-Q MS) equipped with an Agilent HP-5MS column (30 m×0.25 mmi.d., 0.25 m film thickness). The pyrolysis was carried out at 550° C.The chromatograph was programmed from 50° C. (1 min) to 300° C. at arate of 30° C./min; the final temperature was held for 10 min. Heliumwas used as the carrier gas at a constant flow rate of 1 mL/min. Themass spectrometer was operated in scan mode and the ion source wasmaintained at 300° C. The compounds were identified by comparing theirmass spectra with those of the NIST library and those previouslyreported (Del Río J C, Gutierrez A. (2006) J Agric Food Chem54(13):4600-4610; Ralph J, Hatfield R D (1991) J Agric Food Chem39(8):1426-1437). Peak molar areas were calculated for the lignindegradation products, the summed areas were normalized. Analyses on allsamples were conducted in duplicate and data were averaged and expressedas percentages.

In Vivo HCT Activity Assay

For the cloning of AtHCT, total Arabidopsis RNA (1 μg) were extractedusing the Plant RNeasy extraction kit (Qiagen, Valencia, Calif.) andreverse-transcribed using the Transcriptor First Strand cDNA SynthesisKit (Roche Applied Science, Indianapolis, Ind.). The obtained cDNApreparation was used to amplify AtHCT (GenBank accession numberNP_199704.1) using the following oligonucleotides 5′-GGG GAC AAG TTT GTACAA AAA AGC AGG CTT C ATGAAAATTA ACATCAGAGA TTCC-3′ (SEQ ID NO:50; attB1site underlined) and 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGGGTCTCATATCTCAAACAAAAACTTCTCAAAC-3′ (SEQ ID NO:51; attB2 site underlined)for cloning into the Gateway pDONR221-fl entry vector by BPrecombination (Life Technologies, Foster City, Calif.). Asequence-verified AtHCT entry clone was LR recombined with thepDRf1-4CL5-GW vector (41) to generate the pDRf1-4CL5-AtHCT construct.

For HCT activity assays, the pDRf1-4CL5-AtHCT and pDRf1-4CL5 vectorswere transformed into the S. cerevisiae pad1 knockout (MATa his3Δ1leu2Δ0 met15Δ0 ura3Δ0 Δpad1, ATCC 4005833) as previously described (41).Overnight cultures from single colonies harboring the pDRf1-4CL5-AtHCTand pDRf1-4CL5 vectors were grown in 2× yeast nitrogen base mediumwithout amino acids (Difco, Detroit, Mich.) supplemented with 6% glucoseand 2× dropout mix without uracil (Sunrise Science Products, San Diego,Calif.). Overnight cultures were used to inoculated 10 mL of freshminimal medium at an OD₆₀₀=0.1. Substrates (p-coumarate, catechol orbenzoates) were added to the medium 4 h later at a final concentrationof 1 mM and the cultures were grown for 22 h. For the detection of thecoumarate conjugate products, an aliquot of the culture medium wascollected, cleared by centrifugation (20,000×g for 5 min at 4° C.),mixed with an equal volume of 50% (v/v) methanol water and filteredusing Amicon Ultra centrifugal filters (3,000 Da MW cutoff regeneratedcellulose membrane; Millipore, Billerica, Mass.) prior to HPLC-ESI-TOFMS analysis.

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ILLUSTRATIVE SEQUENCES SEQ ID NO: 1-MtAroK polynucleotide sequenceATGGCACCAAAAGCTGTTTTAGTGGGACTTCCTGGAAGTGGAAAGTCCACTATCGGTAGAAGGTTGGCTAAAGCATTAGGAGTTGGTTTGTTAGACACTGATGTGGCTATAGAACAAAGGACAGGAAGATCAATAGCAGACATTTTTGCTACAGATGGTGAACAGGAGTTCAGAAGGATAGAAGAGGATGTTGTGAGAGCTGCATTGGCTGACCATGATGGTGTTCTTAGTTTGGGTGGAGGTGCAGTTACTTCCCCAGGAGTGAGAGCTGCACTTGCTGGTCACACAGTTGTGTATTTGGAAATCTCAGCTGCAGAGGGAGTGAGAAGGACAGGTGGTAACACCGTGAGACCACTTTTGGCAGGTCCTGATAGGGCTGAAAAGTATAGAGCTTTGATGGCAAAAAGGGCTCCTTTATACAGAAGGGTTGCTACTATGAGAGTGGATACAAATAGAAGGAACCCAGGTGCAGTTGTTAGGCACATTTTATCCAGGTTGCAGGTTCCATCTCCTTCTGAGGCAGCTACTSEQ ID NO: 2-MtAroK amino acid sequence (Mycobacterium tuberculosis shikimatekinase; NP_217055)MAPKAVLVGLPGSGKSTIGRRLAKALGVGLLDTDVAIEQRTGRSIADIFATDGEQEFRRIEEDVVRAALADHDGVLSLGGGAVTSPGVRAALAGHTVVYLEISAAEGVRRTGGNTVRPLLAGPDRAEKYRALMAKRAPLYRRVATMRVDTNRRNPGAVVRHILSRLQVPSPSEAATSEQ ID NO: 3-ScAro1 polynucleotide sequenceATGGTTCAGCTTGCTAAGGTGCCTATTTTGGGTAACGACATCATTCACGTTGGATATAACATTCACGATCATTTGGTTGAGACTATTATCAAGCATTGTCCATCTTCTACTTATGTTATTTGTAACGATACCAACCTTTCTAAGGTTCCTTATTACCAACAGTTAGTGCTTGAGTTTAAGGCTTCTTTGCCAGAAGGAAGTAGATTGTTAACTTATGTTGTGAAACCTGGAGAGACTTCTAAGTCAAGGGAAACAAAAGCTCAATTGGAGGACTACCTTTTGGTTGAAGGATGTACCAGAGATACTGTGATGGTTGCTATTGGTGGAGGTGTTATAGGTGATATGATTGGATTTGTGGCATCAACTTTCATGAGAGGTGTTAGGGTTGTGCAAGTGCCAACAAGTTTACTTGCTATGGTTGACAGTTCCATCGGAGGAAAGACAGCAATAGATACCCCATTGGGAAAAAACTTTATTGGTGCTTTCTGGCAGCCTAAGTTCGTGCTTGTTGATATCAAGTGGCTTGAGACATTGGCTAAGAGAGAATTTATCAACGGAATGGCAGAAGTTATCAAGACAGCTTGTATTTGGAACGCAGATGAGTTTACCAGATTGGAATCAAATGCTAGTTTGTTCTTAAACGTTGTGAACGGTGCAAAGAACGTGAAGGTTACTAACCAACTTACAAACGAGATCGATGAAATCTCAAATACCGACATCGAAGCTATGCTTGATCACACTTACAAACTTGTTTTGGAGTCTATCAAGGTGAAAGCAGAAGTTGTGTCTTCAGATGAGAGAGAAAGTTCCTTGAGGAACTTGCTTAACTTCGGTCATTCAATCGGACACGCTTACGAAGCAATCTTAACTCCACAAGCTCTTCATGGAGAATGTGTTTCTATTGGTATGGTGAAGGAGGCAGAATTGTCAAGATACTTCGGAATATTAAGTCCTACACAGGTTGCAAGGTTGTCCAAAATTTTGGTTGCTTACGGTTTGCCAGTGTCTCCTGATGAGAAGTGGTTCAAGGAATTAACACTTCATAAAAAGACCCCTTTAGACATCCTTTTGAAAAAGATGTCCATCGATAAAAAGAATGAGGGTTCTAAAAAGAAAGTTGTGATCTTAGAATCTATCGGAAAGTGCTATGGAGACTCCGCTCAATTTGTTTCTGATGAGGACCTTAGATTCATTTTGACAGATGAAACCCTTGTTTACCCATTTAAAGATATACCTGCTGACCAACAGAAGGTTGTGATTCCACCTGGTAGTAAATCCATTTCTAACAGAGCATTGATCTTAGCTGCATTGGGTGAAGGACAGTGTAAGATAAAGAACCTTCTTCATTCAGATGACACTAAGCACATGCTTACAGCAGTTCATGAATTGAAAGGTGCTACAATCTCTTGGGAGGATAACGGAGAAACCGTTGTGGTTGAAGGTCATGGAGGTTCCACTTTGTCTGCTTGCGCAGATCCACTTTATTTGGGTAATGCTGGAACCGCATCAAGATTTTTAACTAGTCTTGCTGCTTTGGTTAACTCAACTTCTTCACAAAAGTACATTGTGTTAACTGGTAATGCAAGAATGCAACAGAGGCCAATCGCTCCTTTAGTTGATTCTCTTAGAGCAAACGGAACAAAGATCGAGTACCTTAACAACGAAGGTTCACTTCCTATCAAGGTTTACACTGATAGTGTGTTCAAAGGAGGTAGAATAGAATTAGCTGCAACAGTTAGTTCCCAATATGTGTCTTCAATTCTTATGTGTGCTCCATACGCAGAAGAGCCTGTTACTTTAGCTCTTGTGGGAGGAAAGCCAATCTCAAAATTGTACGTTGATATGACAATCAAGATGATGGAAAAGTTCGGAATCAACGTTGAGACTTCTACTACAGAACCATACACATACTACATCCCTAAGGGTCATTACATCAACCCTTCAGAGTACGTTATCGAAAGTGATGCTAGTTCCGCAACTTATCCATTAGCTTTCGCTGCAATGACCGGAACCACTGTGACTGTTCCTAATATTGGATTTGAATCTCTTCAAGGTGACGCTAGATTCGCAAGGGATGTTTTGAAGCCAATGGGTTGTAAAATCACTCAGACAGCTACCTCAACAACCGTTAGTGGTCCACCTGTGGGAACATTAAAGCCACTTAAACACGTTGACATGGAACCTATGACAGATGCTTTCTTGACCGCATGTGTGGTTGCTGCAATTTCACATGATAGTGACCCAAATTCTGCTAACACTACAACCATAGAGGGAATAGCAAACCAAAGAGTTAAGGAATGCAACAGGATCTTGGCTATGGCAACTGAGTTAGCTAAATTTGGTGTTAAAACTACAGAATTACCTGATGGAATCCAGGTGCACGGTCTTAATTCAATCAAGGACTTGAAAGTTCCAAGTGATTCTTCAGGTCCTGTGGGAGTTTGTACTTATGATGACCATAGAGTGGCAATGTCATTCAGTTTGTTAGCTGGTATGGTTAATTCTCAAAACGAGAGGGATGAAGTGGCTAACCCAGTTAGAATTTTGGAAAGGCACTGCACTGGAAAGACATGGCCTGGTTGGTGGGACGTTTTGCATAGTGAATTAGGAGCTAAACTTGATGGTGCAGAGCCTTTAGAATGTACTTCTAAGAAAAATTCCAAGAAATCTGTGGTTATTATCGGAATGAGAGCTGCAGGTAAAACCACTATTTCCAAATGGTGCGCTTCTGCATTGGGATACAAATTGGTTGATTTAGACGAGCTTTTTGAACAACAGCATAATAACCAATCAGTTAAGCAGTTCGTGGTTGAGAACGGTTGGGAAAAATTTAGAGAAGAGGAAACTAGGATCTTCAAGGAAGTTATCCAAAACTACGGTGATGACGGATACGTTTTCTCTACAGGAGGTGGAATTGTGGAGTCAGCTGAAAGTAGAAAGGCACTTAAAGATTTCGCTAGTTCCGGTGGATATGTGTTGCATTTACACAGGGACATTGAGGAAACTATCGTTTTCTTGCAATCTGATCCATCAAGACCAGCTTATGTTGAGGAAATTAGAGAAGTGTGGAACAGAAGGGAGGGTTGGTACAAGGAATGTTCAAACTTCTCTTTCTTTGCTCCACACTGCTCTGCTGAGGCAGAATTTCAAGCTCTTAGAAGGTCCTTCTCTAAATACATCGCAACTATAACAGGAGTTAGAGAGATCGAAATACCATCCGGTAGGTCTGCTTTTGTTTGTTTGACCTTCGATGACTTAACCGAGCAGACTGAAAACTTAACTCCTATTTGTTATGGTTGCGAGGCAGTGGAAGTTAGAGTGGACCATCTTGCTAATTACTCAGCAGATTTCGTTTCCAAGCAATTGTCTATCCTTAGAAAGGCTACTGATAGTATCCCAATAATTTTCACAGTTAGGACCATGAAACAGGGTGGAAACTTTCCTGACGAGGAATTTAAGACACTTAGAGAATTGTACGATATAGCTCTTAAGAATGGTGTTGAGTTTCTTGACTTGGAATTAACTCTTCCTACAGATATCCAATACGAAGTTATCAACAAGAGAGGAAACACTAAGATCATAGGTTCCCATCACGATTTTCAAGGATTATACTCTTGGGATGACGCTGAGTGGGAAAATAGATTCAACCAGGCATTGACCTTAGATGTTGACGTGGTTAAGTTTGTGGGTACTGCTGTTAATTTCGAGGACAACCTTAGATTGGAACATTTTAGGGATACACACAAGAACAAGCCACTTATCGCAGTTAACATGACCTCAAAAGGATCAATCAGTAGAGTGTTGAATAACGTTTTAACCCCTGTGACTTCCGATCTTTTGCCAAACTCTGCTGCACCTGGTCAACTTACCGTTGCTCAGATCAACAAGATGTACACTTCTATGGGTGGAATTGAGCCAAAAGAACTTTTCGTGGTTGGAAAGCCAATCGGACATTCAAGATCACCTATCTTGCATAACACTGGATACGAAATTTTAGGTCTTCCTCATAAGTTCGATAAATTCGAGACAGAATCTGCTCAATTGGTTAAGGAAAAATTACTTGATGGTAACAAGAACTTTGGTGGAGCTGCAGTTACTATCCCATTGAAATTGGATATCATGCAGTACATGGATGAATTGACAGACGCTGCAAAGGTTATTGGTGCTGTGAATACCGTTATCCCACTTGGAAACAAGAAGTTCAAGGGTGATAACACAGACTGGCTTGGAATAAGAAATGCTCTTATCAACAACGGTGTTCCTGAATATGTGGGTCACACTGCAGGATTGGTTATTGGTGCTGGTGGAACATCAAGAGCTGCATTATACGCTCTTCATAGTTTGGGTTGTAAGAAAATCTTTATCATCAACAGGACAACCTCTAAGTTAAAACCACTTATCGAGTCACTTCCTAGTGAATTTAACATCATCGGAATAGAGTCCACTAAGTCTATTGAGGAAATCAAAGAACACGTTGGTGTGGCAGTTTCCTGCGTTCCAGCTGATAAACCTTTGGATGACGAGTTGCTTTCAAAACTTGAAAGATTTTTGGTTAAGGGTGCTCATGCTGCATTCGTGCCAACACTTTTGGAAGCTGCATATAAGCCATCCGTGACCCCTGTTATGACTATCTCTCAGGATAAGTACCAGTGGCACGTGGTTCCTGGATCTCAAATGTTGGTTCATCAGGGTGTGGCTCAGTTTGAGAAGTGGACAGGATTCAAAGGACCATTTAAGGCTATTTTCGACGCAGTTACCAAGGAGSEQ ID NO: 4-ScArol amino acid sequence (Saccharomyces cerevisiae Pentafunctionalarom protein; CAA88208)MVQLAKVPILGNDIIHVGYNIHDHLVETIIKHCPSSTYVICNDTNLSKVPYYQQLVLEFKASLPEGSRLLTYVVKPGETSKSRETKAQLEDYLLVEGCTRDTVMVAIGGGVIGDMIGFVASTFMRGVRVVQVPTSLLAMVDSSIGGKTAIDTPLGKNFIGAFWQPKFVLVDIKWLETLAKREFINGMAEVIKTACIWNADEFTRLESNASLFLNVVNGAKNVKVTNQLTNEIDEISNTDIEAMLDHTYKLVLESIKVKAEVVSSDERESSLRNLLNFGHSIGHAYEAILTPQALHGECVSIGMVKEAELSRYFGILSPTQVARLSKILVAYGLPVSPDEKWFKELTLHKKTPLDILLKKMSIDKKNEGSKKKVVILESIGKCYGDSAQFVSDEDLRFILTDETLVYPFKDIPADQQKVVIPPGSKSISNRALILAALGEGQCKIKNLLHSDDTKHMLTAVHELKGATISWEDNGETVVVEGHGGSTLSACADPLYLGNAGTASRFLTSLAALVNSTSSQKYIVLTGNARMQQRPIAPLVDSLRANGTKIEYLNNEGSLPIKVYTDSVFKGGRIELAATVSSQYVSSILMCAPYAEEPVTLALVGGKPISKLYVDMTIKKMEKFGINVETSTTEPYTYYIPKGHYINPSEYVIESDASSATYPLAFAAMTGTTVTVPNIGFESLQGDARFARDVLKPMGCKITQTATSTTVSGPPVGTLKPLKHVDMEPMTDAFLTACVVAAISHDSDPNSANTTTIEGIANQRVKECNRILAMATELAKFGVKTTELPDGIQVHGLNSIKDLKVPSDSSGPVGVCTYDDHRVAMSFSLLAGMVNSQNERDEVANPVRILERHCTGKTWPGWWDVLHSELGAKLDGAEPLECTSKKNSKKSVVIIGMRAAGKTTISKWCASALGYKLVDLDELFEQQHNNQSVKQFVVENGWEKFREEETRIFKEVIQNYGDDGYVFSTGGGIVESAESRKALKDFASSGGYVLHLHRDIEETIVFLQSDPSRPAYVEEIREVWNRREGWYKECSNFSFFAPHCSAEAEFQALRRSFSKYIATITGVREIEIPSGRSAFVCLTFDDLTEQTENLTPICYGCEAVEVRVDHLANYSADFVSKQLSILRKATDSIPIIFTVRTMKQGGNFPDEEFKTLRELYDIALKNGVEFLDLELTLPTDIQYEVINKRGNTKIIGSHHDFQGLYSWDDAEWENRFNQALTLDVDVVKFVGTAVNFEDNLRLEHFRDTHKNKPLIAVNMTSKGSISRVLNNVLTPVTSDLLPNSAAPGQLTVAQINKMYTSMGGIEPKELFVVGKPIGHSRSPILHNTGYEILGLPHKFDKFETESAQLVKEKLLDGNKNFGGAAVIIPLKLDIMQYMDELTDAAKVIGAVNTVIPLGNKKFKGDNTDWLGIRNALINNGVPEYVGHTAGLVIGAGGTSRAALYALHSLGCKKIFIINRTTSKLKPLIESLPSEFNIIGIESTKSIEEIKEHVGVAVSCVPADKPLDDELLSKLERFLVKGAHAAFVPTLLEAAYKPSVTPVMTISQDKYQWHVVPGSQMLVHQGVAQFEKWTGFKGPFKAIFDAVTKE SEQ ID NO: 5-CgQsuB polynucleotide sequenceATGAGAACAAGTATTGCAACCGTTTGTTTATCCGGAACTCTTGCTGAAAAATTGAGAGCAGCTGCAGACGCAGGATTCGATGGTGTTGAGATTTTTGAACAAGATTTGGTTGTGTCTCCACATTCAGCTGAACAAATCAGACAGAGGGCACAAGATTTAGGTCTTACATTGGACTTATTTCAGCCTTTCAGAGATTTTGAAGGAGTTGAAGAGGAACAATTCTTAAAGAATCTTCACAGGTTGGAGGAAAAATTTAAGTTAATGAACAGACTTGGTATCGAAATGATCTTGCTTTGTTCTAACGTTGGAACAGCTACCATCAACGATGACGATCTTTTTGTGGAACAATTGCATAGAGCTGCAGATTTGGCTGAGAAGTACAACGTTAAGATCGCTTATGAAGCTCTTGCTTGGGGTAAATTCGTTAATGATTTTGAGCATGCTCACGCATTGGTTGAAAAAGTGAACCATAAGGCTTTGGGTACTTGCTTAGATACATTCCACATATTAAGTAGAGGATGGGAGACTGATGAGGTTGAAAACATCCCAGCTGAAAAAATATTTTTCGTGCAATTGGCTGATGCACCTAAGTTATCTATGGATATCCTTTCTTGGTCAAGGCATCACAGAGTTTTTCCAGGAGAGGGTGACTTCGATTTGGTTAAGTTCATGGTGCATCTTGCTAAGACAGGATACGATGGTCCTATATCTTTGGAGATTTTCAACGACTCATTTAGGAAAGCTGAAGTTGGAAGAACTGCAATTGATGGTTTAAGGTCTCTTAGATGGTTGGAGGACCAAACATGGCATGCACTTAACGCTGAAGATAGGCCATCAGCACTTGAGTTGAGAGCTTTGCCAGAAGTTGCAGAGCCTGAGGGTGTGGATTTCATTGAGATCGCTACAGGAAGGTTAGGTGAAACCATCAGAGTTTTACACCAGCTTGGTTTTAGACTTGGTGGACATCACTGTTCTAAGCAGGATTATCAAGTTTGGACTCAAGGAGATGTGAGGATCGTTGTGTGCGACAGAGGAGCAACAGGTGCTCCTACCACTATATCAGCTATGGGTTTCGACACCCCAGATCCTGAGGCTGCACATGCTAGGGCAGAACTTTTGAGAGCACAAACAATTGATAGACCACACATCGAGGGAGAAGTTGATCTTAAGGGTGTGTACGCTCCTGACGGAGTTGAATTGTTTTTCGCAGGACCATCTCCTGATGGTATGCCAGAGTGGTTACCTGAATTTGGTGTTGAGAAGCAAGAAGCTGGACTTATCGAAGCAATCGATCATGTTAACTTTGCTCAGCCTTGGCAACACTTCGATGAGGCAGTTTTGTTTTATACCGCATTGATGGCTTTAGAAACTGTGAGAGAGGATGAATTTCCATCACCTATTGGTTTAGTTAGGAATCAGGTGATGAGATCACCAAACGATGCTGTTAGATTACTTTTGTCAGTGGCACCTGAGGACGGAGAACAGGGTGATTTCTTAAATGCTGCATACCCAGAACATATAGCTCTTGCAACTGCTGATATTGTTGCAGTGGCTGAAAGAGCTAGGAAAAGAGGTTTGGATTTCTTGCCAGTTCCTGAAAACTATTACGACGATGTGCAGGCTAGATTCGATTTGCCTCAAGAGTTTTTAGACACACTTAAGGAAAACCATCTTCTTTATGACTGCGATGAGAACGGTGAATTTTTGCACTTCTACACTAGAACATTGGGAACATTATTTTTCGAGGTTGTGGAAAGAAGGGGTGGATTTGCTGGATGGGGTGAAACCAATGCACCTGTTAGGCTTGCTGCTCAATATAGAGAAGTTAGAGATTTAGAGAGAGGTATCCCAAACSEQ ID NO: 6-CgQsuB amino acid sequence (Corynebacterium glutamicumdehydroshikimate dehydratase; BAF53460)MRTSIATVCLSGTLAEKLRAAADAGFDGVEIFEQDLVVSPHSAEQIRQRAQDLGLTLDLFQPFRDFEGVEEEQFLKNLHRLEEKFKLMNRLGIEMILLCSNVGTATINDDDLFVEQLHRAADLAEKYNVKIAYEALAWGKFVNDFEHAHALVEKVNHKALGTCLDTFHILSRGWETDEVENIPAEKIFFVQLADAPKLSMDILSWSRHHRVFPGEGDFDLVKFMVHLAKTGYDGPISLEIFNDSFRKAEVGRTAIDGLRSLRWLEDQTWHALNAEDRPSALELRALPEVAEPEGVDFIEIATGRLGETIRVLHQLGFRLGGHHCSKQDYQVWTQGDVRIVVCDRGATGAPTTISAMGFDTPDPEAAHARAELLRAQTIDRPHIEGEVDLKGVYAPDGVELFFAGPSPDGMPEWLPEFGVEKQEAGLIEAIDHVNFAQPWQHFDEAVLFYTALMALETVREDEFPSPIGLVRNQVMRSPNDAVRLLLSVAPEDGEQGDFLNAAYPEHIALATADIVAVAERARKRGLDFLPVPENYYDDVQARFDLPQEFLDTLKENHLLYDCDENGEFLHFYTRTLGTLFFEVVERRGGFAGWGETNAPVRLAAQYREVRDLERGIPN SEQ ID NO: 7-PaDsDH polynucleotide sequenceATGCCTTCAAAACTTGCTATCACCTCAATGTCTCTTGGTAGATGCTATGCTGGTCACTCCTTCACTACTAAATTGGATATGGCTAGGAAATATGGTTACCAAGGACTTGAATTGTTCCATGAGGATTTGGCTGACGTTGCATATAGACTTAGTGGTGAAACACCATCCCCTTGTGGACCATCTCCTGCTGCACAGTTGAGTGCTGCAAGACAAATACTTAGGATGTGTCAGGTTAGAAACATAGAAATTGTGTGCTTACAGCCATTTTCTCAATACGATGGTTTGTTAGACAGAGAAGAGCATGAAAGAAGGCTTGAACAATTGGAGTTCTGGATAGAATTAGCTCACGAGCTTGATACAGACATTATCCAGATTCCAGCAAATTTTCTTCCTGCTGAAGAGGTTACCGAAGATATTTCTTTGATCGTTTCAGATTTGCAAGAGGTGGCTGACATGGGTTTGCAGGCAAACCCACCTATTAGATTCGTTTATGAAGCTCTTTGTTGGTCAACTAGAGTGGATACATGGGAAAGGAGTTGGGAGGTTGTGCAAAGAGTTAATAGGCCTAACTTTGGTGTGTGCCTTGATACATTCAATATCGCAGGAAGAGTTTACGCTGACCCAACCGTGGCATCAGGTAGAACTCCTAACGCTGAAGAGGCAATTAGGAAGTCAATCGCTAGATTGGTTGAAAGGGTTGATGTTAGTAAAGTTTTCTATGTGCAAGTTGTGGACGCAGAGAAGTTGAAAAAGCCATTAGTTCCTGGACACAGATTCTACGATCCAGAACAACCTGCTAGGATGTCTTGGTCAAGAAACTGCAGGTTGTTTTATGGTGAAAAAGATAGAGGAGCTTACTTGCCAGTTAAGGAGATTGCTTGGGCATTTTTCAATGGTTTGGGATTTGAAGGTTGGGTTTCCTTAGAGCTTTTCAACAGAAGGATGTCTGATACTGGTTTTGGAGTGCCTGAAGAGTTAGCTAGAAGGGGAGCAGTTTCCTGGGCTAAACTTGTGAGAGATATGAAGATCACCGTTGACTCACCAACTCAACAGCAAGCTACACAGCAACCTATAAGAATGTTGAGTTTATCCGCTGCATTASEQ ID NO: 8-PaDsDH amino acid sequence (Podospora anserina dehydroshikimatedehydratase; CAD60599)MPSKLAITSMSLGRCYAGHSFTTKLDMARKYGYQGLELFHEDLADVAYRLSGETPSPCGPSPAAQLSAARQILRMCQVRNIEIVCLQPFSQYDGLLDREEHERRLEQLEFWIELAHELDTDIIQIPANFLPAEEVTEDISLIVSDLQEVADMGLQANPPIRFVYEALCWSTRVDTWERSWEVVQRVNRPNFGVCLDTFNIAGRVYADPTVASGRTPNAEEAIRKSIARLVERVDVSKVFYVQVVDAEKLKKPLVPGHRFYDPEQPARMSWSRNCRLFYGEKDRGAYLPVKEIAWAFFNGLGFEGWVSLELFNRRMSDTGFGVPEELARRGAVSWAKLVRDMKITVDSPTQQQATQQPIRMLSLSAAL SEQ ID NO: 9-PhPAAS polynucleotide sequenceATGGACACTATCAAGATCAACCCAGAGTTCGACGGACAGTTCTGCAAGACTACATCATTATTAGACCCAGAGGAGTTCAGGAGGAATGGACATATGATGGTTGATTTTCTTGCTGACTACTTCCACAACATCGAAAAGTACCCAGTTAGATCCCAAGTGGAACCTGGTTATTTGGAGAGGTTGTTACCAGATTCAGCTCCTATACAGCCAGAACCTATCGAGAAAATTTTGAAGGATGTTAGATCAGACATATTTCCAGGTTTAACACATTGGCAAAGTCCAAATTTCTTTGCTTACTTCCCTTGCTCTTCAAGTACCGCAGGAATTTTAGGTGAAATGCTTTCAGCTGGATTGAACGTTGTGGGTTTTTCATGGATCGCTAGTCCAGCTGCAACTGAATTAGAGAGTATTGTTATGGATTGGCTTGGAAAATTGATTAATTTGCCTAAGACATATCTTTTCTCTGGTGGAGGTGGAGGTGTGATGCAGGGTACTACATGCGAAGTTATGCTTTGTACTATCGTGGCTGCAAGAGATAAAATGTTGGAAAAGTTTGGAAGGGAGAACATTGATAAGTTAGTTGTGTACGCATCAGACCAAACCCACTTTAGTTTCCAGAAAGCTGTTAAGATCTCAGGTATAAAACCAGAAAACTTCAGAGCTATACCTACCACTAAGGCAACAGAATTCTCCCTTAACCCAGAGTCTTTGAGAAGGGCTATCCAAGAGGATAAAAAGGCAGGACTTATCCCTTTGTTTTTATGCACATCAATAGGTACAACCAGTACTACAGCAGTTGACCCACTTAAACCTTTGTGTGAAATAGCTGAAGAGTATGGAATTTGGGTTCATGTGGATGCTGCATACGCTGGTTCTGCATGCATTTGTCCTGAATTTCAGCATTTCTTGGACGGTGTTGAGCACGCTAATTCCTTTTCTTTCAACGCACACAAGTGGTTGTTTACTACTCTTGATTGTTGCTGTCTTTGGTTGAAAGACCCATCCTCTTTGACTAAGGCACTTTCAACAAACCCTGAAGTTTTGAGAAACGATGCTACCGACAGTGAGCAAGTTGTGGATTATAAAGACTGGCAGATTACTTTATCCAGAAGGTTTAGGTCTCTTAAGCTTTGGTTGGTTCTTAAGTCCTACGGAGTGGCTAATCTTAGAAACTTCATAAGGTCTCATATCGAAATGGCTAAGCACTTTGAAGAGTTGGTTGCAATGGATGAAAGATTCGAGATCATGGCACCAAGGAATTTTTCCTTAGTTTGTTTCAGAGTGTCTCTTTTGGCTCTTGAAAAGAAGTTTAATTTCGTTGATGAAACTCAAGTGAACGAGTTTAACGCTAAGCTTCTTGAATCTATCATCTCAAGTGGTAACGTTTACATGACACATACCGTTGTGGAGGGAGTTTACATGATTAGATTCGCTGTGGGTGCACCTTTGACAGATTATCCTCACATTGATATGGCTTGGAATGTTGTTAGGAACCACGCTACTATGATGTTGAACGCASEQ ID NO: 10-PhPAAS amino acid sequence (Petunia hybrida Phenylacetaldehydesynthase; ABB72475)MDTIKINPEFDGQFCKTTSLLDPEEFRRNGHMMVDFLADYFHNIEKYPVRSQVEPGYLERLLPDSAPIQPEPIEKILKDVRSDIFPGLTHWQSPNFFAYFPCSSSTAGILGEMLSAGLNVVGFSWIASPAATELESIVMDWLGKLINLPKTYLFSGGGGGVMQGTTCEVMLCTIVAARDKMLEKFGRENIDKLVVYASDQTHFSFQKAVKISGIKPENFRAIPTTKATEFSLNPESLRRAIQEDKKAGLIPLFLCTSIGTTSTTAVDPLKPLCEIAEEYGIWVHVDAAYAGSACICPEFQHFLDGVEHANSFSFNAHKWLFTTLDCCCLWLKDPSSLTKALSTNPEVLRNDATDSEQVVDYKDWQITLSRRFRSLKLWLVLKSYGVANLRNFIRSHIEMAKHFEELVAMDERFEIMAPRNFSLVCFRVSLLALEKKFNFVDETQVNEFNAKLLESIISSGNVYMTHTVVEGVYMIRFAVGAPLTDYPHIDMAWNVVRNHATMMLNASEQ ID NO: 11-ObCCMT1 polynucleotide sequenceATGGCGAGAAAAGAGAACTATGTTGTTTCTAACATGAATGTTGAAAGTGTGTTGTGCATGAAAGGTGGAAAAGGAGAAGATAGCTATGATAACAACTCTAAGATGCAGGAGCAACATGCTCGATCAGTGCTCCACCTTCTGATGGAAGCTCTCGACGGCGTGGGGCTGAGCTCGGTGGCGGCCGGCGCTTTCGTGGTGGCGGATCTCGGCTGCTCCAGCGGAAGAAACGCCATAAACACGATGGAATTTATGATCAATCACCTGACTGAGCACTACACGGTGGCGGCGGAAGAGCCGCCGGAATTCTCAGCCTTCTTCTGCGACCTCCCCTCCAACGACTTCAACACCCTCTTTCAGCTCCTTCCGCCGTCTGACGGCAGCAGCGGTTCTTACTTCACTGCCGGCGTGGCCGGTTCGTTTTACCGGAGGCTTTTCCCGGCGAAGTCTGTTGATTTCTTTTACTCGGCATTTAGTTTGCACTGGCTATCTCAGATACCAAAGGAGGTGATGGAGAAGGGATCGGCGGCTTACAACGAGGGGAGAGTGACCATCAACGGTGCAAAAGAGAGCACCGTAAATGCATACAAGAAACAATTTCAAAGTGATTTGGGTGTCTTCTTGAGATCCAGATCCAAAGAATTGAAACCGGGAGGATCCATGTTCCTCATGCTCTTGGGTCGGACCAGCCCCGACCCGGCAGATCAGGGCGCATGGATTCTCACTTTCAGCACACGTTATCAAGATGCTTGGAATGATCTTGTGCAAGAGGGCTTAATTTCGAGCGAAAAACGGGATACGTTCAACATCCCGATATATACGCCCAGCCTAGAGGAGTTCAAAGAGGTGGTAGAAAGAGATGGTGCATTCATAATCAACAAGCTCCAACTTTTCCACGGTGGCAGCGCTCTCATCATCGATGATCCCAACGATGCGGTTGAGATTAGCCGTGCCTATGTCAGCCTCTGTCGCAGCCTCACCGGAGGCTTAGTTGATGCCCACATAGGCGATCAGCTCGGCCATGAGCTCTTCTCGCGCTTATTAAGCCAAGCCGTGGATCAGGCTAAGGAGCTAATGGACCAGTTTCAGCTCGTCCATATAGTTGCATCCCTTACTTTAGCTSEQ ID NO: 12-ObCCMT1 amino acid sequence (Ocimum basilicum cinnamate/p-coumaratecarboxyl methyltransferases; ABV91100)MARKENYVVSNMNVESVLCMKGGKGEDSYDNNSKMQEQHARSVLHLLMEALDGVGLSSVAAGAFVVADLGCSSGRNAINTMEFMINHLTEHYTVAAEEPPEFSAFFCDLPSNDFNTLFQLLPPSDGSSGSYFTAGVAGSFYRRLFPAKSVDFFYSAFSLHWLSQIPKEVMEKGSAAYNEGRVTINGAKESTVNAYKKQFQSDLGVFLRSRSKELKPGGSMFLMLLGRTSPDPADQGAWILTFSTRYQDAWNDLVQEGLISSEKRDTFNIPIYTPSLEEFKEVVERDGAFIINKLQLFHGGSALIIDDPNDAVEISRAYVSLCRSLTGGLVDAHIGDQLGHELFSRLLSQAVDQAKELMDQFQLVHIVASLTLA SEQ ID NO: 13-RgC2′H polynucleotide sequenceATGGCACCAACCAAAGATTCAGTTATTCACATGGGAGCAGAGTCCTGGGATGAGATTTCCGAGTTCGTTACTAAAAAGGGACACGGTGTTAAGGGTCTTTCTGAACTTGGTATTAAAACTCTTCCAAAGCAATTCCATCAGCCTCTTGAAGAGAGGTTCAGTGAGAAAAAGATTTTGGAAAGAGCTTCAATCCCACTTATCGATATGAGTAAGTGGGACTCCCCTGAGGTTGTGAAGTCTATCTGTGATGCTGCAGAACATTGGGGTTTCTTTCAAATAGTTAATCACGGAGTGCCATTGGAGACTTTACAGAGAGTTAAAGAAGCTACACATAGGTTTTTCGCTTTGCCTGCAGAAGAGAAAAATAAGTACTCTAAGGAAAACTCACCAATTAATAACGTTAGATTCGGTTCTTCATTCGTTCCTCATGTTGAGAAAGCACTTGAATGGAAGGATTTTCTTAGTATGTTCTATGTTTCCGAAGAGGAAACTAACACATACTGGCCACCTATTTGTAGAGACGAGATGTTAGAATACATGAGGAGTTCCGAGGTTCTTATCAAAAGATTGATGGAAGTGTTAGTTGTGAAGGGTCTTAAAGTTAAGCAAATCGATGAGATAAGAGAACCAATGTTGGTGGGATCAAGAAGAATTAATTTGAACTACTACCCTAAATGCCCAAATCCTGAACTTACATTGGGTGTTGGAAGGCATAGTGATATTTCCACCTTTACTATCTTGTTACAAGACGAAATCGGTGGACTTCATGTTAGAAAGTTGGATGACACTGGTAACACCTGGGTTCATGTTACCCCAATATCTGGTTCACTTATTATCAATATCGGAGATGCTTTGCAGATAATGTCTAACGGAAGGTACAAGTCAATAGAACACATGGTTGTGGCAAATGGAACACAAGACAGAATCTCTGTTCCTTTATTTGTGAACCCAAAGCCTCAGGCTATACTTTGTCCATTCCCTGAGGTTTTGGCAAATGGAGAAAAACCAGTTTATAAGCCTGTGTTGTGCTCTGATTACTCAAGGCATTTCTACACAAAACCTCACGATGGTAAAAAGACAGTGGATTTCGCATTGATGAACSEQ ID NO: 14-RgC2′H amino acid sequence (Ruta graveolens 2-oxoglutarate-dependentdioxygenase; Vialart et al. plant J 2012, 70: 460-470)MAPTKDSVIHMGAESWDEISEFVTKKGHGVKGLSELGIKTLPKQFHQPLEERFSEKKILERASIPLIDMSKWDSPEVVKSICDAAEHWGFFQIVNHGVPLETLQRVKEATHRFFALPAEEKNKYSKENSPINNVRFGSSFVPHVEKALEWKDFLSMFYVSEEETNTYWPPICRDEMLEYMRSSEVLIKRLMEVLVVKGLKVKQIDEIREPMLVGSRRINLNYYPKCPNPELTLGVGRHSDISIFTILLQDEIGGLHVRKLDDTGNTWVHVTPISGSLIINIGDALQIMSNGRYKSIEHMVVANGTQDRISVPLFVNPKPQAILCPFPEVLANGEKPVYKPVLCSDYSRHFYTKPHDGKKTVDFALMNSEQ ID NO: 15-Plastid targeting signal polynucleotide sequenceATGGCTTCGATCTCCTCCTCAGTCGCGACCGTTAGCCGGACCGCCCCTGCTCAGGCCAACATGGTGGCTCCGTTCACCGGCCTTAAGTCCAACGCCGCCTTCCCCACCACCAAGAAGGCTAACGACTTCTCCACCCTTCCCAGCAACGGTGGAAGAGTTCAATGCATGCAGGTGTGGCCGGCCTACGGCAACAAGAAGTTCGAGACGCTGTCGTACCTGCCGCCGCTGTCGACGATGGCGCCCACCGTGATGATGGCCTCGTCGGCCACCGCCGTCGCTCCGTTCCAGGGGCTCAAGTCCACCGCCAGCCTCCCCGTCGCCCGCCGCTCCTCCAGAAGCCTCGGCAACGTCAGCAACGGCGGAAGGATCCGGTGCATGCAGSEQ ID NO: 16-Plastid targeting signal amino acid sequenceMASISSSVATVSRTAPAQANMVAPFTGLKSNAAFPTTKKANDFSTLPSNGGRVQCMQVWPAYGNKKFETLSYLPPLSTMAPTVMMASSATAVAPFQGLKSTASLPVARRSSRSLGNVSNGGRIRCMQSEQ ID NO: 17-IRX5 promoter polynucleotide sequenceATGAAGCCATCCTCTACCTCGGAAAAACTTGTTGCGAGAAGAAGACATGCGATGGCATGGATGCTTGGATCTTTGACATTGATGACACTCTTCTCTCAACCATTCCTTACCACAAGAGCAACGGTTGTTTCGGGTAAATAAACTAAACTTAACCATATACATTAGCCTTGATTCGGTTTTTGGTTTGATTTATGGATATTAAAGATCCGAATTATATTTGAACAAAAAAAAATGATTATGTCACATAAAAAAAAATTGGCTTGAATTTTGGTTTAGATGGGTTTAAATGTCTACCTCTAATCATTTCATTTGTTTTCTGGTTAGCTTTAATTCGGTTTAGAATGAAACCGGGATTGACATGTTACATTGATTTGAAACAGTGGTGAGCAACTGAACACGACCAAGTTCGAGGAATGGCAAAATTCGGGCAAGGCACCAGCGGTTCCACACATGGTGAAGTTGTACCATGAGATCAGAGAGAGAGGTTTCAAGATCTTTTTGATCTCTTCTCGTAAAGAGTATCTCAGATCTGCCACCGTCGAAAATCTTATTGAAGCCGGTTACCACAGCTGGTCTAACCTCCTTCTGAGGTTCGAATCATATTTAATAACCGCATTAAACCGAAATTTAAATTCTAATTTCACCAAATCAAAAAGTAAAACTAGAACACTTCAGATAAATTTTGTCGTTCTGTTGACTTCATTTATTCTCTAAACACAAAGAACTATAGACCATAATCGAAATAAAAACCCTAAAAACCAAATTTATCTATTTAAAACAAACATTAGCTATTTGAGTTTCTTTTAGGTAAGTTATTTAAGGTTTTGGAGACTTTAAGATGTTTTCAGCATTTATGGTTGTGTCATTAATTTGTTTAGTTTAGTAAAGAAAGAAAAGATAGTAATTAAAGAGTTGGTTGTGAAATCATATTTAAAACATTAATAGGTATTTATGTCTAATTTGGGGACAAAATAGTGGAATTCTTTATCATATCTAGCTAGTTCTTATCGAGTTTGAACTCGGGTTATGATTATGTTACATGCATTGGTCCATATAAATCTATGAGCAATCAATATAATTCGAGCATTTTGGTATAACATAATGAGCCAAGTATAACAAAAGTATCAAACCTATGCAGGGGAGAAGATGATGAAAAGAAGAGTGTGAGCCAATACAAAGCAGATTTGAGGACATGGCTTACAAGTCTTGGGTACAGAGTTTGGGGAGTGATGGGTGCACAATGGAACAGCTTCTCTGGTTGTCCAGTTCCCAAGAGAACCTTCAAGCTCCCTAACTCCATCTACTATGTCGCCTGATTAAATCTTATTTACTAACAAAACAATAAGATCAGAGTTTCATTCTGATTCTTGAGTCTTTTTTTTCTCTCTCCCTCTTTTCATTTCTGGTTTATATAACCAATTCAAATGCTTATGATCCATGCATGAACCATGATCATCTTTGTGTTTTTTTTTCCTTCTGTATTACCATTTTGGGCCTTTGTGAAATTGATTTTGGGCTTTTGTTATATAATCTCCTCTTTCTCTTTCTCTACCTGATTGGATTCAAGAACATAGCCAGATTTGGTAAAGTTTATAAGATACAAAATATTAAGTAAGACTAAAGTAGAAATACATAATAACTTGAAAGCTACTCTAAGTTATACAAATTCTAAAGAACTCAAAAGAATAACAAACAGTAGAAGTTGGAAGCTCAAGCAATTAAATTATATAAAAACACTAACTACACTGAGCTGTCTCCTTCTTCCACCAAATCTTGTTGCTGTCTCTTGAAGCTTTCTTATGACACAAACCTTAGACCCAATTTCACTCACAGTTTGGTACAACCTCAGTTTTCTTCACAACAAATTCAAACATCTTACCCTTATATTACCTCTTTATCTCTTCAATCATCAAAACACATAGTCACATACATTTCTCTACCCCACCTTCTGCTCTGCTTCCGAGAGCTCAGTGTACCTCGCCSEQ ID NO: 18-AtC4H promoter polynucleotide sequenceCGGAATGAGAGACGAGAGCAATGTGCTAAGAGAAGAGATTGGGAAGAGAGAAGAGAAGATAAAGGAAACGGAAAAGCATATGGAGGAGCTTCACATGGAGCAAGTGAGGCTGAGAAGACGGTCAAGTGAGCTTACGGAAGAAGTGGAAAGGACGAGAGTGTCTGCATCGGAAATGGCTGAGCAGAAAAGAGAAGCTATAAGACAGCTTTGTATGTCTCTTGACCATTACAGAGATGGGTACGACAGACTTTGGAGAGTTGTTGCAGGACATAAGAGTAAGAGAGTAGTGGTCTTATCAACTTGAAGTGTAAGAACAATGAGTCAATGACTACGTGCAGGACATTGGACATACCGTGTGTTCTTTTGGATTGAAATGTTGTTTCGAAGGGCTGTTAGTTGATGTTGAAAATAGGTTGAAGTTGAATAATGCATGTTGATATAGTAAATATCAATGGTAATATTTTCTCATTTCCCAAAACTCAAATGATATCATTTAACTATAAACTAACGTAAACTGTTGACAATACACTTATGGTTAAAAATTTGGAGTCTTGTTTTAGTATACGTATCACCACCGCACGGTTTCAAAACCACATAATTGTAAATGTTATTGGAAAATAGAACTCGCAATACGTATTGTATTTTGGTAAACATAGCTCTAAGCCTCTAATATATAAGCTCTCAACAATTCTGGCTAATGGTCCCAAGTAAGAAAAGCCCATGTATTGTAAGGTCATGATCTCAAAAACGAGGGTGAGGTGGAATACTAACATGAGGAGAAAGTAAGGTGACAAATTTTTGGGGCAATAGTGGTGGATATGGTGGGGAGGTAGGTAGCATCATTTCTCCAAGTCGCTGTCTTTCGTGGTAATGGTAGGTGTGTCTCTCTTTATATTATTTATTACTACTCATTGTAAATTTCTTTTTTCTACAATTTGTTTCTGACTCCAAAATACGTCACAAATATAATACTAGGCAAATAATTATTTTATTATAAGTCAATAGAGTGGTTGTTGTAAAATTGATTTTTTGATATTGAAAGAGTTCATGGACGGATGTGTATGCGCCAAATGGTAAGCCCTTGTACTGTGCCGCGCGTATATTTTAACCACCACTAGTTGTTTCTCTTTTTCAAAAAACACAAAAAAAAAATAATTTGTTTTCTTAACGGCGTCAAATCTGACGGCGTCTCAATACGTTCAATTTTTTTCTTTCTTTCACATGGTTTCTCATAGCTTTGCATTGACCATAGGTAAAGGGATAAGGATAATGGTTTTTTCTCTTGTTTGTTTTATCCTTATTATTCAAAAAGGATAAAAAAACAGTGATATTTAGATTTCTTTGATTAAAAAAGTCATTGAAATTCATATTTGATTTTTTGCTAAATGTCAACACAGAGACACAAACGTAATGCACTGTCGCCAATATTCATGGATCATGACAATAAATATCACTAGAATAATTAAAAATCAGTAGAATGCAAACAAAGCATTTTCTAAGTAAAACAGTCTTTTATATTCACGTAATTGGAATTTCCTTTTTTTTTTTTTGTCGTAATTGGAATTTCCTTTATCAAACCCAAAGTCCAAAACAATCGGCAATGTTTTGCAAAATGTTCAAAACTATTGGCGGGTTGGTCTATCCGAATTGAAGATCTTTTCTCCATATGATAGACCAACGAAATTCGGCATACGTGTTTTTTTTTTTGTTTTGAAAACCCTTTAAACAACCTTAATTCAAAATACTAATGTAACTTTATTGAACGTGCATCTAAAAATTTTGAACTTTGCTTTTGAGAAATAATCAATGTACCAATAAAGAAGATGTAGTACATACATTATAATTAAATACAAAAAAGGAATCACCATATAGTACATGGTAGACAATGAAAAACTTTAAAACATATACAATCAATAATACTCTTTGTGCATAACTTTTTTTGTCGTCTCGAGTTTATATTTGAGTACTTATACAAACTATTAGATTACAAACTGTGCTCAGATACATTAAGTTAATCTTATATACAAGAGCACTCGAGTGTTGTCCTTAAGTTAATCTTAAGATATCTTGAGGTAAATAGAAATAGTTGACTCGTTTTTATCTTCTTCTTTTTTTACCATGAGCAAAAAAGATGAAATAAGTTCAAAACGTGACGAATCTATATGTTACTACTTAGTATGTGTCAATCATTAAATCGGGAAAACTTCATCATTTCAGGAGTATTACAAAACTCCTAAGAGTGAGAACGACTACATAGTACATATTTTGATAAAAGACTTGAAAACTTGCTAAAACGAATTTGCGAAAATATAATCATACAAGTGCCAGTGATTTTGATCGAATTATTCATAGCTTTGTAGGATGAACTTAATTAAATAATATCTCACAAAAGTATTGACAGTAACCTAGTACTATACTATCTATGTTAGAATATGATTATGATATAATTTATCCCCTCACTTATTCATATGATTTTTGAAGCAACTACTTTCGTTTTTTTAACATTTTCTTTTGTTGGTTATTGTTAATGAGCATATTTAGTCGTTTCTTAATTCCACTGAAATAGAAAATACAAAGAGAACTTTAGTTAATAGATATGAACATAATCTCACATCCTCCTCCTACCTTCACCAAACACTTTTACATACACTTTGTGGTCTTTCTTTACCTACCACCATCAACAACAACACCAAGCCCCACTCACACACACGCAATCACGTTAAATTTAACGCCGTTTATTATCTCATCATTCACCAACTCCCACGTACCTAACGCCGTTTACCTTTTGCCGTTGGTCCTCATTTCTCAAACCAACCAAACCTCTCCCTCTTATAAAATCCTCTCTCCCTTCTTTATTTCTTCCTCAGCAGCTTCTTCTGCTTTCAATTACTCTCGCCSEQ ID NO: 19-AtC3H promoter polynucleotide sequenceATCGTAAGTTTTTTTGTGTGTGTGTTAACAATGTACTCACTACTCACTGTTCCATATTTTTGATGTACGTATATCGAAAACATTCTGCCAACAAATGCAAACATAACAAAAGTCAAAAACAATAACATAACCGGGAATTAAACCAAAATGTAATTGCTTTTTATTAGTGTCAGGCCTTCTGCTTAAAAATATTCTCGGCCCAGAGCCCATTAACACCTATCTCAATTCATATTGAAGAAAATGACTATATTACTTGACAAAAACTTTAGTCAGAAAAATATGGAATCTCTTTCGGTACTGCTAAGTGCTAACCTTAAATAGTATAGAATTCTTAGTTCATTCTCAAAAACATAGCTATATGTAGATTATAAAAGTTCGATATTATTTCCTGCAAAAGATGTTATAATGTTACAACTTACAAGAAAATGATGTATATGTAGATTTTATAAACTGGTACCGTAATTCATAAAAGATGGTGGTGGGTATGTATCAGTAACGGAACTTACATATGCGTGTGTATTACTATGTCTATATGGTGTATTCCTTTGTGTGGAACAATGCACGTCAGAGTTGTTTATTTTCTTATAGAATTTAAGGAATCAATTATTGGATTTCTCAAGGTGAAAGTGGACTTCTTTGCACGCAAGGTCTAGTTGCCGACTTGCCGTTGCATGTAACATGATTGTTGAAATAAAGTGAATTGAGAGAAGTTTGGCCAGACATTTTAAATTTAACCCAAAAAAAGTAGGGCCTAACACAAAATATAACCTCTCTTTGTTCAAAGGAAATAACACCTACGTCTTATAATTGAACCAAACATTGAATCATTGAACTCACCTATAATAATTATAATAACACGAATTCACAAGACACCTAAAAGAAAAAGTTCACAAAAACAAATAAAAATTTACCTCTCACCAAACACACTCACCTACCCGTCTGGTCCCACTGACCCCAACATACAACACCGACTCTCTCCCACACCAATTTTTTTTTTTGGCGTTTTAAAACAAATAAACTATCTATTTTTTTTTCTTACCAACTGATTAATTCGTGAATAATCTATTATCTTCTTCTTTTTTTTGTGACGGATGATTAGTGCGTGGGGAAATCAAAATTTACAAAATTTGGGATGATTCCGATTTTTGCCATTCGATTAATTTTGGTTAAAAGATATACTATTCATTCACCAAGTTTTCAGATGAGTCTAAAAGATAATATCATTTCACTAGTCACTTAAAAAAAGGGTTAAAAGAACATCAATAATATCACTGGTTTCCTTAGGTGACCCAAAAAAAGAAGAAAAAGTCACTAGTTTCTTTTTGGAAATTTTACTGGGCATATAGACGAAGTTGTAATGAGTGAGTTTAAATTTATCTATGGCACGCAGCTACGTCTGGTCGGACTATACCAAGTTACCAACTCTCTCTACTTCATGTGATTGCCAATAAAAGGTGACGTCTCTCTCTCTCTCACCAACCCCAAACCACTTTCCCCACTCGCTCTCAAAACGCTTGCCACCCAAATCTATGGCTTACGGGGACATGTATTAACATATATCACTGAGTGAAAAGAAGGGTTTATTACCGTTGGACCAGTGATCAAACGTGTTTTATAAAAATTTGGAATTGAAAACATGATTTGACATTTTTAATGATGGCAGCAGACGAAACCAACAACACTAAGTTTAACGTTCGTGGAGTATACTTTTCTATTTTCGAAGAAGACATATAACTAAGCTGATTGTTATTCTTCATAGATTTCTTTTCACTGCGAATAAAAGTTTGTGAACATGTCACCGTTTGAACACTCAACAATCATAAGCGTTTTACCTTTGTGGGGTGGAGAAGATGACAATGAGAAAGTCGTCGTACATATAATTTAAGAAAATACTATTCTGACTCTGGAACGTGTAAATAATTATCTAAACAGATTGCGAATGTTCTCTACTTTTTTTTTGTTTACATTAAAAATGCAAATTTTATAACATTTTACATCGCGTAAATATTCCTGTTTTATCTATAATTAATGAAAGCTACTGAAAAAAAACATCCAGGTCAGGTACATGTATTTCACCTCAACTTAGTAAATAACCAGTAAAATCCAAAGTAATTACCTTTTCTCTGGAAATTTTCCTCAGTAGTTTATACCAGTCAAATTAAAACCTCAAATCTGAATGTTGAAAATTTGATATCCAAGAAATTTTCTCATTGGAATAAAAGTTCAATCTGAAAATAGATATTTCTCTACCTCTGTTTTTTTTTTTCTCCACCAACTTTCCCCTACTTATCACTATCAATAATCGACATTATCCATCTTTTTTATTGTCTTGAACTTTGCAATTTAATTGCATACTAGTTTCTTGTTTTACATAAAAGAAGTTTGGTGGTAGCAAATATATATGTCTGAAATTGATTATTTAAAAACAAAAAAAGATAAATCGGTTCACCAACCCCCTCCCTAATATAAATCAAAGTCTCCACCACATATATCTAGAAGAATTCTACAAGTGAATTCGATTTACACTTTTTTTTGTCCTTTTTTATTAATAAATCACTGACCCGAAAATAAAAATAGAAGCAAAACTTCSEQ ID NO: 20-AtHCT promoter polynucleotide sequenceTTCTCTAGGTTTTGAAGCTTTCCTAGTTCTTTTGGAAGCGTGCCGGACAAGTCATTGTCGTATAGAAACAGATTGATAAGTTCAGAGCAGTTTCCAAGCTCTTTAGGGATCTCACCTGAGAGCATTGTAGAATAGACAGATAAAGACTGGAGCTTGCTTAGTTGACCCAACGAAACAGGTAAAGAACCGGATATTTTCGTTGCTGCTAACCCTAAGACCTTGAGATTCCTACAGTTTCCGATCTCCTCCGGGATCTTCCCTGAAAGCTCTGAGTTTCCTCCGGCTCTTATGCTCTCAAGAGTCGAGATCTTTCCGAGCTCCAACGGGAGATTCTCGGATAAGTAGTTATCGAAAATCTCAAGATTCTTGAGGCTAACGCAGTCGCCGAGTTCCGGTGGGATCTTTCCTGTGAGGCCATTGGAGTTTAAACAAAGTTCTTGAAGATTCTTGAGCTTCCCTAGACTCGAAGGTATTTCACCAACAAGACTATTTGAGCTTAAATCGATAACTATAAGCTCCGAACAATCTCCGATCTCAGAAGATATAGCTCCGGTGAGATTAGTGTTGGAGATAACGAGTTTCTGAAGTGAAGTAAACGAAGAAATGTTAGGAGGGAAAGGTAAAGCTAACTGAACAGAGACGACATTGATCTCTGTAACGAGTTTGTTGTCTGAGGAGGAACAAGTAATGTAAGGCCATTGACATGGGTCAGAATCAGAAGGATTCCAGCCGGAGAAGACTGACGGTGGCGGCGAGTTCGAGCTGTGAAGCCAAGAAATCAAAGCTGAGACTTCATTGGTTGATGCAGAGGTCGAGGAGATGAAGAAAGCTAAAAACAGAGACAATGTAATGGAAAAATGAGAAACAGTTAAGGCTTTTTTTCTTGGAATCGGCATTTGCAAAGACATAAGAGTTTTTTTCTTTGCATTTGGCTCTCAAATCCAAAACAAGCCTTCTTGGTTCTGCATCGATCTGAGTCCTTTGGCTTAGGGTTTAGGGAAGTTTTTGCTTTAGAGATAAGCAATAAGAAAGAATGATATATTAAATATATAAAAGTACTAAACTTCATGTGCTCTGTCTTTTTCTTTTACCTCGGGGTTCTGTTTCTAGCTTCAGATTAATTAATTACAGTCATTAACTTTTCTTTGAAATATGTTTGCCAAGAGCCCGAGACACTATCCATAGATGACAAAAGTCAATAGTTATATATACATAAAATATCACAAAACAAAAGGCATTGGTTATATATATACAGAATCATTTCACTTAGTAGTGTTTTTTCTTATAAGATTATGATAGAAATATGGAAGCATGCATGTGGTTTTGCATTGTTTTCCTCAATTAAGTCAGGATTGTGAGTTGGTTTGTTTTCGAGACCTGAACCGAGCGTTTAAGATTCTTCCTCGTTTGAAGTAAACTCCATAATTGTCCACACCTAAGCTAAAAGAAAGTAATAACAAGTTTAAATATTCATGACAAGGAAAATATTGCATTCAGAAAATTGTTAACAACGAAGTAAACATTTTTTTCAATCCGATGCCAATAGTCTCTAGCGGCATCAAAAGTCCACAAACTCGATACCTCTGGGTAAATGAGCGAATGGGCCGGTCCGTTGTAGCCCAGAAGAGAAATTGTCCTCTAAATTCCATACTTCCATGAATTTTCTCTGTATATCCTCGTTTGATGTATGGTATATTTGTTCCGCTCTAAATCATGACCAACCCAAGGTACTAAATTGTCATTTAAGCTTTGATTGGTATTTGGTAGCATGGGTTACCATTGACCAACCCACGGTACTAGTTGCTTTTCTTTTAGTTTTGCTTTTGCTTTATTTTCTTAGAGAGTGGGAGGACAAAAGGTTTGGATCATTAAGCCAATGAATGCTTCAAAGAAATTGAATTTTTATTAGATCCTCAAACCAAGTTGGATCATCAAATAATGGCTAAGAAATAATTTTAGAACAGAAAGCAAAGAAAAGCTATCCGCAACAACAACCATTAGTTAATAAATTAAAATGAAATGTGAAATTTATGACTAATTGAGGTATGTTTTCATATAATATAGTATAGTTCGGATATAAATTCAACATAATTTATTTGTGGTGTACTGAAAAAAAGACTTTCTTGGATTCTGACGTAATTCTCTTAACACGTGAGTTTACGCCGTTAGATGTTATTGGTGGTTGTTGTTATGCTCTGCTACGTGGTAATGAGTTAAGTTAAGCCAAACTTTGGCATTCGATTGACTAACTTGTACGGTAGCTATAACAATCAACTTGTCAATTTTTTTTCCTTCTTCTTCATTCGAACTTTATACTATTTAAGCCCATTAGTATTATTTGGGCCTTAGGACAGAGGGAACGGGTTTACCAACCCCGGATAGAAAAGTAGGACCGAGTGATGAGATGGACCAATGATAAACCTTCTGAGAGAGTTGGTCGACAGATGGAGTAGGCGGGGTCGTGGGGCGGTAGGTGAAGGATTACGACCTTTCCTTTTTTGTTCACACCCACTTATATCTACCCCTCCTCGCTTCTCACACAATTTCTCAGATCAAACTCAAAACAAAATTTGTTTGTTCGTTTGATCTTTCTTAAAAATSEQ ID NO: 21-AtCCR1 promoter polynucleotide sequenceTTGCTTTCTCTGTCCATGATATGAGGCATTGACTTCTCACCTGTATTCATATGGTATAGATTCCTCTTTTCAGGAGTCCAATACAAACGAGCTTGGTGAAGAACTCGTTGGTAAGAGAGTTAATGTCTGGTGGCCACTCGACAAGAAGTAAGTTTATTGTTAAACTTACTAACTTCATTTTTGATACTATATGATGAATGATAGCAATCTTACGATTTGTATTTGCACAGGTTTTATGAAGGTGTCATAAAATCTTATTGTAGAGTTAAGAAGATGCATCAAGTGAGTTAACTTCTCTATTTGGTATTTTAAAATTCTCTATTTATTGCATAACTGGTTTATATAGAATTTTCCCACTGATGGTCTCGCAGGTAACATATTCTGATGGCGATGTTGAAGAGCTTAATCTGAAAAAAGAACGTTTTAAGATAATCGAGGATAAATCTTCAGCCAGTGAGGTGAAAATTTCTTACATTCTATCATTCACCATTCTTTATATTTACCAAAATTTCAATGTATCTGGTTTCCCTAATAAAATCTAAGCAGGATAAGGAAGATGATCTGCTTGAGTCTACTCCTTTATCTGCCTTGTAAGTGAAACTTCCATAGTTCTATGATAACCCACAATTTATAATTTTAATTTAGCTTTAGTCTTGAGTTTTTTGCTGTTATGTGCAGTATACAAAGGGAGAAATCCAAGAAGAGGAAAATTGTGTCTAAGAATGTGGAACCGAGTAGTTCTCCAGAAGTCAGGTATGAAAGTATATAAGAATTCTAGTTTTAGTTGTTTGAAAGTTTGATCCGTGAGTGAATTAGTTCACAATTATGGATGTAGATCCTCTATGCAAACAATGAAGAAGAAAGACTCTGTAACAGACTCCATTAAGCAAACAAAAAGAACCAAAGGTGCACTGAAGGCTGTAAGCAATGAACCAGAAAGCACTACAGGGAAAAATCTTAAATCCTTGAAAAAGCTGAATGGTGAACCTGATAAAACAAGAGGCAGAACTGGCAAAAAGCAGAAGGTGACTCAAGCTATGCACCGGAAAATCGAAAAAGATTGTGATGAGCAGGAAGACCTCGAAACCAAAGATGAAGAAGACAGTCTGAAATTGGGGAAAGAATCAGATGCAGAGCCTGATCGTATGGAAGATCACCAAGAATTGCCTGAAAATCACAATGTAGAAACCAAAACTGATGGAGAAGAGCAGGAGGCAGCGAAAGAGCCAACGGCAGAGTCTAAAACTAATGGAGAGGAGCCAAATGCAGAACCCGAAACTGATGGAAAAGAGCATAAATCATTGAAGGAGCCAAATGCAGAGCCCAAATCTGATGGAGAAGAGCAGGAGGCAGCAAAAGAGCCAAATGCTGAGCTCAAAACTGATGGAGAAAATCAGGAGGCAGCAAAAGAGCTAACTGCAGAACGCAAAACTGATGAGGAAGAGCACAAGGTAGCTGATGAGGTAGAGCAAAAGTCACAGAAAGAGACAAATGTAGAACCGGAAGCTGAGGGAGAAGAGCAAAAGTCAGTGGAAGAGCCAAATGCAGAACCCAAGACCAAGGTAGAAGAGAAAGAGTCAGCAAAAGAGCAAACTGCAGACACAAAATTGATTGAGAAGGAGGATATGTCTAAGACAAAGGGAGAAGAGATTGATAAAGAAACATATTCAAGCATCCCTGAGACTGGTAAAGTAGGAAACGAAGCTGAAGAAGATGATCAGAGAGTGATTAAGGAACTGGAAGAAGAGTCTGACAAGGCAGAAGTCAGTACTACGGTGCTTGAGGTTGATCCATGAATGAAGGATTGTTAGGTAAATGTTAATCCAGGAAAAAAAGATTGGTTCTTGTGGTTTAGGTAACTTATGTATTAAGTGAAGCTGCTTGTTTAGAGACTAATGGTGTGTTTTATGAGTAGATTCTTCTGACCTATGTCTCGTTATGGAACTAGTTTGATCTTATGTCACCTTGCTAGCAGCAGATATTGATATTTATATATTTAAGAGACATGCGCATGAGAATGAGGGTATGGAAAAGTCCATATCAGATGACACAAACAATGATCGTATGTGTAGTCACTTGTGCATTTCCAGTTTTGGACATAAAATTCTGATATTGCATAGAAATGTTTTTAAATAACACTAATCCAAACCTAAATAAAATATCTCTATACATCATCTAGAAATGTATGGCTTGATCAAGAATTGTAGATAATAATACCCTGAGTTAAATGATTGTAGGTATTATTTCAGTTTTCAAAATTGTCCAAATTTATGAGCTATATTAAAGATAATATTTTCAATAAGGTGTGTAGTTCTAAATGTTTCTTCTTCTTCCACCAACCCCTCTTTCTATATGTATGTTCTTTTTTCTAAAATAATTGTTTGTTCTTTTTTAGATATATCAAATTAAATATAAAAAATATTGACAAAACTTATTTACCATTGTTAGGTGAACTTGGCAAGTGTGTAAATATAAAGATAACATTCCTTTTCGTTCTTTATATATACGAAACGTACCACAAATTTCTAACTAAAGCATTCATAGTCTCTCGAAAGCCTCTTTTCAGAACCGAAGCTCTTTACTTTCGTCCACCGGGAAATSEQ ID NO: 22-AtCAD4 promoter polynucleotide sequenceCAGAAAGGTCTTCACACTCTGTTTTAGCTAGAGAGTTTTATCCATCTGAGTTTTTAGTCTATTTTGTTTTATCTAGGAGTTGCTTTGTTTGTTCGAATTCGGTCATTGCTTTTGCTGCTTTACTGGAGTCAAATTTGAAGGTAAAATATATGTTAAATATCTGGGTAGGTGGTTGTGGATGATGGAAAATCTGAACGTATCACTGTTAATGACAATGGAGAACTCGTTTCTACTCAGCATGCTATCACCGAATACCGAGTGATTGAATCTTCACCACATGGTTAGTGAGACTGACTTCCATTTCTATTCAGTTAAACTTAAAGCAAATGATTTTGCCTTGAGTTTTTAGCACATTGTTGAATTGCAGGATACACATGGCTTGAGCTTCGCCCTTTAACCGGGAGAAAACATCAGGTCTCTATAGATATTCAGTTTTTGTTTCAACTTTCTCTCTTTTTTATGTTCTCTTAATACTAATCTGTTTTCAACTGTTCTTCGATTGCCACAGCTTCGTGTACACTGCGCTGAAGTGCTAGGAACACCGATAGTCGGGGACTACAAATACGGTTGGCAAGCTCATAAAGCCCGGGAACCTTTTGTCTCTTCTGAAAACAACCCAACCAAGCAATCATCATCTCCTTTTGGATTGGATCTGGATGGTGGAGATGTCTCTTCGAAACAGCCACACCTTCATCTCCATTCAAAGCAAATCGATCTGCCAAACATATCACAGCTCTTGGAGAAAATGCAGGTCTCTTCAGACTCTGATATTTCGGATCTCGATAGCCTTAAATTCGATGCTCCATTGCCTAGTCATATGCAACTAAGCTTTAATTTGTTGAAATCTAGAGTCGAAACTTGTGACAAAAATTAGATTTTTTTTCTTACCGAGCTTTCTTCTTTGTGTTCATTGAGGCCCAAGTATTTGTGTATTTGGACCTGAATATTCTCATACAAAGATAAATAATTATAATTAAATGATTTTTCGCATATAATCATTATTGTGGTATGATTAACACAGTTGGTGTGATGACTGATTGACACAATAATCACCGTTTGGATTCGATTCCTTTAATACTTGTCACTAGAGTTGTTTGACTAAACAGCTAACTTGTCACTAGAGTTATTGTGTTTGTATTTTGATCTGTTATTAATCTGATTGGGTATAATTACAGATAGAGAGACATCTATATTGTAATTAAGACAATCTTAAAGTGTAAACTAAAAAGATCTCTCTGACCTCTGGAAAACGAAAGGTGGGTGACACATCACTCTAGCTATGAATATGATGAATATTCAGTACCTAACCGAACAAAGACTGGTTTGGTATTTTTATTGGAAAAAAGAGATAAATAATTGTGAATGTGAATTATCCTGTCTGAAAGGTAAGCTGATGACATGGCGTTATATGATTGGACGAGCTTCAGAACAAAAGAGTAGCGTCGAATCGAATCTTTACCTACTACACTTTGAACTTTGAAGTACATTACCTACTTCCTCCTTGATCGAACGTCTTTTCTCAAAACTATTTTATTTCCCCAATTAAAGTAGTGGTGATAAATTCACAAAAATACAAACACTTTTATTTTTGACGTCAAAAACAAATACTTCTTTGAACAGGCTATTACAATATTTTTAAGAAAAAAGTAAGCAAAATAGTCCACAAACCAAAATCTGTAACATATTAAACGATTTATGTTTTTTTTTTTTTTTCTTAACTAGAGAACAATTCGGGCTTTTACTAAGGATGATGAGTGTAGTTACCGAATAGTGTATTCATATAATCTTTTAATGAGCTTAAGATATGATATTATTTCGACTAATCAGATAAGAGTAGTTAGATAATTTCGTAATAGAGCAACTCTTTCGCAAATAAAACCATTGTAAACATTACCAATTAGTTTTTCTTTTTTTTTGGTCACAACCAATTAGTTTGTTTGTTCTATTTTATGAAGTGCGTATTAAAGCTAACGTGTTTACAGTAACGCCACACAAATAAAAATAAAAATAATTATGTACTTTATGGATTTATAGAAAAAACAAGAATAGTCACCAAAAATTGATTGTGTCATATATCTTTTGTCAACTATTTTATCTTATTTTTCTATGGATATGTATGTCCAAAATGTTAGACAAAAAACCAAAAAATCATGTCCAAAATTTCGTTAGGCTGCCGATATCTCTGTTTCCCTTTCAACGACTATCTATTTAATTACCGTCGTCCACATTGTTTTTAATATCTTTATTCGAGGTTGGTTTAGTTTTTTTTACCAAACTCACTTTGCTACGTTTTTGCCTTTTTGGTATGGTTGTATTTGTACCACCGGGAAAAAAAAGATAAGAGGTTTGGTTGGTCGAGCTTACTGATTAAAAAATATACACGTCCACCAAATATTAAAACAATATATCCCATTTTTCCTCCTCTCTTTTGGTATTACATTAATATTTTATTATTTCCCCATTTGCTCTGTATATATAAACATATGTCAATAGAGTGCCTCTACAGTCATGTTTCCATAGACATAATCTCTCACCATTGTTTTTCTCTGCAAAACTAAAGAAACAAAAAAAGAAAAATCGGAGAAACCAAGAAAAAAGAASEQ ID NO: 23-AtCAD5 promoter polynucleotide sequenceCCTCGATAACTCTGATTGTTGTATTGTCCAAGTATTCACTAAACAACTTTGCTAAAAGAGAAGATGCTGCTGGAGCAATTTCAGAAGGTTTTAGCACAACCGCATTACCAGCTGCAATAGCTCCAATGACTGGCTCGACAGACAATACTAAGGAAAAAAACAAAGCACCATGAAGACATATAAACTTTAATAGTTTAGAAATTGAGACAAAATTGTCAATAAATAAAATTGAGCTTACAGAAAGGGAAATTCCAGGCTGAAATAACCAAAACAACTCCAAGCGGTTCTGAGACTATTTGTGCAGACGAGGGAAATGTTGTCACAGAAGTTTTGACCTGAAAGGTCCAAGCATAGAAAAAGCAAGTGGTTTTAGAAAGGACACATATCAATGAAGCAGCAAAGCTTGAACGGTCTAGTTACCGTTTCTGGAGCCATCCAGTTCTTTAPCTCTTTGATTGCAPGCATACAGGATGATTTTGTATTCGAAATCTAAAAAACGAGAAAAATACCAAAGAGATTCAACAGTGGATAAGTGGAATGCAGTGAAGAAACGGGACATTGAAATTATATAAAAAACCTCAGCTAGAAAAGCTTCAAGCTCAGGCTTAGAAAGATCTTGATACAAAGCTTCGGTGATGCATTTCTCCTTCTCATCAATCATCCTAGCAATGTTTTGAAGCTGAGAAATTCTCCACTCGTAGCTCTTCGTTCTGCCAGAGTTGAAGTTGCTTCTGAGCTCATCTACAAGCAAAGCTGCTTCTTTTCCACTAAAGTCTGATGCTTGCTCCTTTACCACAGCAGATAGTGTTGCATAACAAGTACTGATTCAAGACACCAAAACCGCAATGTGAGAGACTTTAAGACTAAAAATCATGGATAAGACTAAAAAAACATGGATAAGTATCAACTGTTCTCACGATTATTTATTCATACCACTGTACTTAAACTTAAAACCCACTATACTAAATAGAAAGGTAATCATCAAAAAATCAGTATGTAAAAACCACTTTTGTGAATAAAATATGTAAAATGGGTGAATAAAGAAATGTGCTTACAATTTCAACCGATAAGGGATACAAGCATTGCTGCAATATCCACCACCACCACGACGAGATATCCGAAAAGGTGAAGTTGCAACATTTAATCTGCAACAAAAGAGGCCATTCATTAAAATGGTACTAATTAGATCTAATCATATCATATTGAATGACCAAATCATTCACAGAAGCATCCATTGCTCCAATTAACATTCTAGACCAAATTCAACTTAAAGGTAACTCTTTTATACAGGAAACCGAGAAACCGAAAACGCAATTCACATAAAAAGGAAGGCTTGTTTGGAGAAGCAGAATCGAACAAGTCAATCTCAAACCCTGATGAGCAGGTTTTTCAAGTTACCTGGCAGGAGAAAAACCCTTGGCAAAACAAAGGGTTTGAATATGATTAATCTCTAGAAGCTTCGTCATGACTTGGGTTCAGTTAAAAATCTCAAATTGGAGACATTATTGGTGTTTATATATTTGAGAGAGAGAGCCAGAGAGGAGACGTTGAATTGAATGAAGGGTGTGGTCGGAAGAGAAGACGTGTAGAAGAGACGAGACAAGTAAATTTAAGCATTGGCCCCATTTACAGCCACAAGTCCGCTACAACAAATTATTTCCAAGAAACTCTGAGATAACGTCGTGATGAAACGGCTCATGCTGCTGTTGTGATTCGTGAATTAGAGGTTTATCTTTTGGGTTTTTGAATGTTACTTAATTGGACGGTCGATTTTTCAAACTGGGTGTGAAATGTGAATGGGTCATTCATAATGGGCTTTTGTTTTAATGTGAAGCCATTCACACACTCTTTGTCCTTCTTTTCTATTATTCATAACTGTCACTCTTTGTTCTTCGAAATAGTAAAGAGCAAATCGATTCTTTGTTGATCTGGGCCGTAAAATTTCCATGGTTGTGGGAAGTATTCTCGCAGCTGATCTGGGCCGTCAATGCTACAGTTTCATGTCAGAGAGAGGTCAAGAATCAACACGTGGCCAACCATGATTTTAAACCAAAGCAAACACACGATTAGACCCCACATTGTTTGTTCACCAACCCCCGTGGACCCTCCTTTAGCCGACGTGTCCACGTCAATAGTGGTTTTTCTTCCTTTCAAAGTACACAAATTCCATTCTTTCTCATTTTACTTTTTGGATTACGTTGTTGTTATAAACTGGTAAAATGAATTATGAATGCAAATAAATTTCATTTAAGTTTTGTTGGCTTCTAATATTTTTTTCACCTAAAATTCTAATAAACTACACAGCCATGAGCCATCGTATGAAAAGAAGAAGAAAAAAAATGTCTTTTTCTAGAAGGATCTTTCAACGACTAAAAAAGATTTTAAGCTTTTGACTAATTTTGTCAATAATATACACAAATTTACACTCAATTATAGCCATCAAATGTGTGCTATGCAGAAACACCAATTATTTCATCACACATACGCATACGTTACGTTTCCAACTTTCTCTATATATATATATAGTAATACACACACATAAACAGCAAAAGCGTGAAAGCAGCAGATCAAGATAAGAAAGAAGAAAGAATCATCAAAAASEQ ID NO: 24-AtF5H promoter polynucleotide sequenceTGTGTGTCTTTTTGCGAGTAGTTGTTGGCTTCAGACAGTTCATAGCGGAGTTACTCTATACGCGAPGTACTTGTCTCATACTGATAATTTTGATGGCAATTAAGGCTTTAAAAGCTTATGTATTTTCTTATAACCATTTTATTCTGTATATAGGGGGACAGAAACATAATAAGTAACAAATAGTGGTTTTATTTTTTTAAATATACAAAAACTGTTTAACCATTTTATTTCTTGGTTAGCAAAATTTTGATATATTCTTAAGAAACTAATATTTTAGGTTGATATATTGCAGTCACTAAATAGTTTTAAAAGACACGAAGTTGGTAAGAACAGGCATATATTATTCGATTTAATTAGGAATGCTTATGTTAATCTGATTCGACTAATTAGAAACGACGATACTATGAGCTCATAGATGGTCCCACGACCCACTCTCCCATTTGATCAATATTCAACTGAGCAATGAAACTAATTAAAAACGTGGTTAGATTAAAAAAATAAATTGTGCAGGTAGCGGATATATAATACTAGTAGGGGTTAAAAATAAAATAAAACACCACAGTATTAAATTTTTGTTTCAAAAGTATTATCAATAGTTTTTTTGCTTCAAAAATATCACAAATTTTTGTATGAAATATTTCTTTAACGAAAATAAATTAAATAAAATTTAAAATTTATATTTGGAGTTCTATTTTTAATTTAGAGTTTTTATTGTTACCACATTTTTTGAATTATTCTAATATTAATTTGTGATATTATTACAAAAAGTAAAAATATGATATTTTAGAATACTATTATCGATATTTGATATTATTGACCTTAGCTTTGTTTGGGTGGAGACATGTGATTATCTTATTACCTTTTTATTCCATGAAACTACAGAGTTCGCCAGGTACCATACATGCACACACCCTCGTGAAACGAGCGTGACTTAATATGATCTAGAACTTAAATAGTACTACTAATTGTGTCATTTGAACTTTCTCCTATGTCGGTTTCACTTCATGTATCGCAGAACAGGTGGAATACAGTGTCCTTGAGTTTCACCCAAATCGGTCCAATTTTGTGATATATATTGCGATACAGACATACAGCCTACAGAGTTTTGTCTTAGCCCACTGGTTGGCAAACGAAATTGTCTTTATTTTTTTATGTTTTGTTGTCAATGTGTCTTTGTTTTTAACTAGATTGAGGTTTAATTTTAATACATTTGTTAGTTTACAGATTATGCAGTGTAATCTGATAATGTAAGTTGAACTGCGTTGGTCAAAGTCTTGTGTAACGCACTGTATCTAAATTGTGAGTAACGACAAAATAATTAAAATTAAAGGGACCTTCAAGTATTATTAGTATCTCTGTCTAAGATGCACAGGTATTCAGTAATAGTAATAAATAATTACTTGTATAATTAATATCTAATTAGTAAACCTTGTGTCTAAACCTAAATGAGCATAAATCCAAAAGCAAAAATCTAAACCTAACTGAAAAAGTCATTACGAAAAAAAGAAAAAAAAAAGAGAAAAAACTACCTGAAAAGTCATGCACAACGTTCATCTTGGCTAAATTTATTTAGTTTATTAAATACAAAAATGGCGAGTTTCTGGAGTTTGTTGAAAATATATTTGTTTAGCCACTTTAGAATTTCTTGTTTTAATTTGTTATTAAGATATATCGAGATAATGCGTTTATATCACCAATATTTTTGCCAAACTAGTCCTATACAGTCATTTTTCAACAGCTATGTTCACTAATTTAAAACCCACTGAAAGTCAATCATGATTCGTCATATTTATATGCTCGAATTCAGTAAAATCCGTTTGGTATACTATTTATTTCGTATAAGTATGTAATTCCACTAGATTTCCTTAAACTAAATTATATATTTACATAATTGTTTTCTTTAAAAGTCTACAACAGTTATTAAGTTATAGGAAATTATTTCTTTTATTTTTTTTTTTTTTTAGGAAATTATTTCTTTTGCAACACATTTGTCGTTTGCAAACTTTTAAAAGAAAATAAATGATTGTTATAATTGATTACATTTCAGTTTATGACAGATTTTTTTTATCTAACCTTTAATGTTTGTTTCCTGTTTTTAGGAAAATCATACCAAAATATATTTGTGATCACAGTAAATCACGGAATAGTTATGACCAAGATTTTCAAAGTAATACTTAGAATCCTATTAAATAAACGAAATTTTAGGAAGAAATAATCAAGATTTTAGGAAACGATTTGAGCAAGGATTTAGAAGATTTGAATCTTTAATTAAATATTTTCATTCCTAAATAATTAATGCTAGTGGCATAATATTGTAAATAAGTTCAAGTACATGATTAATTTGTTAAAATGGTTGAAAAATATATATATGTAGATTTTTTCAAAAGGTATACTAATTATTTTCATATTTTCAAGAAAATATAAGAAATGGTGTGTACATATATGGATGAAGAAATTTAAGTAGATAATACAAAAATGTCAAAAAAAGGGACCACACAATTTGATTATAAAACCTACCTCTCTAATCACATCCCAAAATGGAGAACTTTGCCTCCTGACAACATTTCAGAAAATAATCGAATCCAAAAAAAACACTCAATSEQ ID NO: 25-AtPAL1 promoter polynucleotide sequenceCAAATAGTACGATGTATTTAGTGATTTTATTTATGTACTTTGTTCATTAAATTAGTCATAATTGTTCTGATTTTTAGGGGTTTTGATCGAACCCTTAGATCAAAAGTTACCTTAATTGTTTTTTTAGCTAAGTACTTTATTAAAAATTTAATGTTTAGTTCTGATTGAGTAGTACTATAAAGGAGACATGTGTCAATCTTGTCAATTGGTTTTGAGTTCAACAATATGCAATATTGCACATGCATTAACGACCAAAAGAAGATGCAATGCACTTAAATCATTGAAACTGATTTTGTTTTTGTAGTGTATAAAATATCTATTTAATTACCAACGAAAGAAGTGAGCTTTTAAAAACAAAGAGTCAGAAGATATATATAACTACAAAACCTACAGAAGATAAGCTGGATTTCAAAAGAAGAGAAAGAGTAAACCAATAAATTGACCAAAGCAAAATCGGATATTTGACATAAGTTTCCATTCACATTGACCCAAATCCACCAGCATTTCAAATAAAGTTACTTAATATAATTTTTGTGTTTATAATATATTCCGCCCACTCTTGCCTTCATTTGGACCTTATCCTAAAAGTCAAAACAGGTGAAAAAAATGAGAATACAATTAACACGAAAAATGCAAAAGACTGTTAAACCGAAATCGAATTCTAGTGTAATCAATCCTTTTCCCAATGATACAACTATAAATCAAAAAGAAAAAATGTACTGATAAACGAAACTAAACGTATAAATTAATATATTTCTTGACATAAATAGGAGGCTTTTGCCTGCTAGTCTGCTACGATGGAAGGAAAAATGCATGCACACATGACACATGCAAAATGTTTCAATGAAGACGCATTGCCCAATTAACCAACACACCACTTCTTCCATTCCACCCATATTATTTATTTCTACCATTTTCTTTAATTTATTGTTTTTTCTTTGATTCATACACTGTTTATGACTATTACATTTTCCCTTTCGACTAATATTAACGCGTTTAAACCAAAGAATGGATTTGATAATGAAATTTTATTTTATTAGCATATAGATAATGGATGGCTTCATGCTTGGTTTCCATGACAAGGAATGACACAAGATAATTATTTTGAATAAAATCATAAATATGATAATACTAGTTGTAAAAAAACTTGAGTGTTTCGTGTGTTATTTTTCGGTTTCTTGACTTTTTATATTTCTCGTTTTTGTAATTTTAGGATGGATTATTTAGCTTGCTTTTCTCTTTTATTACTTTCTAAAATTTTATTTATAAACTCATTTTTAATATATTGACAATCAATAAATGAGTTATCTTTTAATTAATAAAAAATTTGTAAACTCTTGTAAACAGATCATAGTCACTAAAAGCTATTATAAGTTATTTGTAGCTATATTTTTTTATTTCATGAACTTAGGATAAGATACGAAAATGGAGGTTATATTTACATAAATGTCACCACATTGCCTTTGTCATGCAAACGGCGTGTTGCGTCACTCGCCTCCTATTGGGAATCTTATAATCGCGTGAATATTATTAGAGTTTGCGATATTTCCACGTAATAGTTATCTTTCACAAATTTTATACTCAATTACAAAATCAACGAAAATGTACATTTGTATCTTTAACTATTTACGTTTTTTTTACGTATCAACTTTCAGTTATATGTTTTGGATAATATATTTTTTTACTTTTGACTTTTCAGTTTTCACCTAATGATTGGGATATACATATGCATGCATAGTTCCCATTATTTAAATGTAAGCTAAGTGCATATGAACTGTTAGTCAAAATTACGAAGTTTATTTGTACATATATATAGTTATAACAAAATGGTACAGTAAATTAAACAGAACATCAAGAAAGTACAAAAGACTGAACACAATAATTTACATGAAAACAAAACACTTAAAAAATCATCCGATAAAATCGAAATGATATCCCAAATGACAAAAATAACAATATAGAAAATACAAAAACAAAAACAAAATATGAAAGAGTGTTATGGTGGGGACGTTAATTGACTCAATTACGTTCATACATTATACACACCTACTCCCATCACAATGAAACGCTTTACTCCAAAAAAAAAAAAAAAACCACTCTTCAAAAAATCTCGTAGTCTCACCAACCGCGAAATGCAACTATCGTCAGCCACCAGCCACGACCACTTTTACCACCGTGACGTTGACGAAAACCAAAGAAATTCACCACCGTGTTAAAATCAAATTAAAAATAACTCTCTTTTTGCGACTTAAACCAAATCCACGAATTATAATCTCCACCACTAAAATCCATCACTCACTCTCCATCTAACGGTCATCATTAATTCTCAACCAACTCCTTCTTTCTCACTAATTTTCATTTTTTCTATAATCTTTATATGGAAGAAAAAAAGAAACTAGCTATCTCTATACGCTTACCTACCAACAAACACTACCACCTTATTTAAACCACCCTTCATTCATCTAATTTTCCTCAGGAACAAATACAATTCCTTAACCAACAATATTACAAATAAGCTCCTATCTTCTTTCTTTCTTTTAGAGATCTTGTAATCTCCTCTTAGTTAATCTTCTATTGTAAAACTAAGATCAAAAGTCTAASEQ ID NO: 26-AtPAL2 promoter polynucleotide sequenceGATTGATGGTTTAATAATCTGCCTCGTGATACATGGTGTTATCTTAAAATGGTCTCTCAATTAGTCTTTGTATTTGTATAAAATAAGGCCTAAAAATATCATCAATGGGGTCCTGTTAAAAACAAAAACAGATACACCTTTCACTAATAAAAAAAAACTGTTACCGACAAGTCAAACAATATCTGCGGACAAAAAAATGAAGAATGTTTAGTAAGAAATAGAAGATGTGGTAAAGAGCCATACACACATGCAAGTGTTTTTCAATGAACCCATCTTACCAACCCACTACTTCTTTGAGCCATAATTGTTTGGTTCGGAGACCCTTTACATTTCCGTCTCAGCTTTATTTGTTTACGCATTGATTTGTCTTAAATTATGTTAGATATTGTTTTTTGGCTATTTATTAGCAGCAATCAAGTTAAAAGAGTGGTTCGATATCACCATCGAACTCTCCTTTAGATATTTTCTATATAAAACCAAACAAAAACAAAAAAATTGGTCCGATCATCTAATATACAAGTTAGACGATTTCACGTTATGTTATTACAACCTACAACAAAATAGACTATGATCGAAATCATATTGAATCTTTTACCTTTCAACGTAATACAAATCTGGCTTTACAAAGCAATAATTCATGTTTGTTTGTCTAATTTAAATTTCCCTGTTTTTTTTCCCCTCTTTCTGTTTCCCATTTGAAAGTAAAAGATCATTTAAGCACCTAACTCAATTTTATTTTATTTTAAACACCTAATGTCATGCTCCTTGGCTCCTTGTAATTAGTTGATCGTTTCAATTTAGACCAGCAAAACATTTTAGTATGTTCGTAAATATTGCGTACATGCCATTTCGTTTGTCATGCAAACGGTGTGTGTTTCTTTACTTAGCTTCTAGTTGGTGTATATTGCGTCGCATTAATATCGGTTTACCTTCCTCCTGTCTACGTAATGATATATTCTCCACCACAAATTTAAATTCTTATTGAAATTTCCTAATTTTTTAGGTAGCTCAAGGTCTCAAGTATACTACGTACCCTATTTTTTTGAATATCTATCTATATTATAACAAGAGTTTTTCTGAGCTAGTTAATGAGATGACAATATTCTACATAAATAAATGACCCTCGAAAGTTTCAAGTACTTTAGGATCTGACCAAATCGGGGTAAAACATTTTGAAACTAATTACGTTCACATCTACCATCGATGATTGACAAGCTTATTGTCACCTTTTATGTTAAAGTGACATGGTCTTGACGTTAATTTGCATGTTATTCTACATCTATAGTCCAAAGATAGCAAACCAAAGAAAAAAATTGTCACAGAGGGTTCAATGTTACTTAGATAGAAATGGTTCTTTACAATAATAAATTTATGTTCCATTCTTCATGGACCGATGGTATATATATGACTATATATATGTTACAAGAAAAACAAAAACTTATATTTTCTAAATATGTCTTCATCCATGTCACTAGCTCATTGTGTATACATTTACTTGCTTCTTTTTGTTCTATTTCATTTCCTCTAACAAATTATTCCTTATATTTTGTGATGTACTGAATTATTATGAAAAAAAACCTTTACACTTGATAGAGAAGCATATTTGGAAACGTATATAATTTGTTTAATTGGAGTCACCAAAATTATACAAATCTTGTAATATCATTAACATAATAGCAAACTAATTAAATATATGTTTTGAGGTCAAATGTTCGGTTTAGTGTTGAAACTGAAAAAAATTATTGGTTAATAAAATTTCAAATAAAAGGACAGGTCTTTCTCACCAAAACAAATTTCAAGTATAGATAAGAAAAATATAATAAGATAAACAATTCATGCTGGTTTGGTTCGACTTCAACTAGTTAGTTGTATAAGAATATATTTTTTTAATACATTTTTTTAGCAACTTTTGTTTTTGATACATATAAACAAATATTCACAATAAAACCAAACTACAAATAGCAACTAAAATAATTTTTTGAAAACGAAATTAGTGGGGACGACCTTGAATTGACTGAACTACATTCCTACGTTCCACAACTACTCCCATTTCATTCCCAAACCATAATCAATCACTCGTATAAACATTTTTGTCTCCAAAAAGTCTCACCAACCGCAAAACGCTTATTAGTTATTACCTTCTCAATTCCTCAGCCACCAGCCACGACTACCTTTTCGATGCTTGAGGTTGATATTTGACGGAACACACAAATTTAACCAAACCAAACCAAAACCAAACGCGTTTTAAATCTAAAAACTAATTGACAAACTCTTTTTGCGACTCAAACCAAATTCACGTTTTCCATTATCCACCATTAGATCACCAATCTTCATCCAACGGTCATCATTAAACTCTCACCCACCCCTCATACTTCACTTTTTTTCTCCAAAAAATCAAAACTTGTGTTCTCTCTTCTCTCTTCTCTTGTCCTTACCTAACAACAACACTAACATTGTCCTTCTTATTTAAACGTCTCTTCTCTCTTCTTCCTCCTCAGAAAACCAAAAACCACCAACAATTCAAACTCTCTCTTTCTCCTTTCACCAAACAATACAAGAGATCTGATCTCATTCACCTAAACACAACTTCTTGAAAACCASEQ ID NO: 27-At4C11 promoter polynucleotide sequenceACATAAGATTTGGATTATGAGAGGAGTTGAGAAGTTATATGATGGAAACTGAAAAGTAAATCTTTTTGCAGAGCTGTAGAATCAATCAACATTTGATGACTTGGACTTCTTCACCATGTGTGTTGGTGTGGACCATTGAATTGACGGTTTTGCCATTCACCAACAACAGCATGAGTTTTTGAGTCTTCATGTTTGGTAAAGGTTAGGCTTATTAGGAGACACGGGTAAGAGACTAGAGAGAGACATTCTCCAAACCTTTCTTTTGCATGTTTTGTAAGAAACATTTCCGAAAATGAAAGAAATCTTACACAACATTCATATAATTTGTTTGAAATATAACAAAATGATAATTTATACTCTCAAGTAAAATGCCTAAACTTTTATCAATTGGAAAAGACATCACACACAAGCGTGAAGCGTATCTTATTACCAAACCCAACTAAGCATGGGTCTCGATACTTGCCATAATTACTTTAATCCATTCTCTTTTTGAGAAATGTATAAAACATGACTTTGCATAAATAGTCTTTTACTAATTACTATGTAAATAATTCCTAAGACTGGTTTCATGGTACATATTATCGTTTTATCCTTGTTTTAAGAATATTCAGATGTTTGGTCTATGGAATATAGTCTATTCTTCATGTTTAAAACTATTATTTGATAAGAAAATATGTACTAATATGTTTTTGCATACAAATGTTGATCAGTTCGTAGCATTTGAATTAATACATTCTCAATCACTTTCAAGCATTATTATGTAATAAATGATTCATGTCGAAAAGTAATAGTATCACTGTCCATTACATTTGGCATATATATTTTTTTGTCAAAGCCTTACATTTGGCATATTGACGAAGCAGTTTTGTATTCACTTATATTTTGACATCGCTTTCACAAAAATAAATAGCTATATATGATTATTATCCATTAATTGTCTCTTTTCTTTTGCTGACACAATTGGTTGTAAATGCAATGCCAATATCCATAGCATTTGTGTGGTGAATCTTTTTCTAAGCCTAATAGTAAATAAATCTCAATACAAGAACCCATTTACGAACAAATCAAACCAAGTTGTGATGGGTTAGTACTTAGTAGCCCGTTTGAAATGTAGAATTTTTGATGAGATTTTACGTTTTATATAGATTTTTCTCAGAAAACAAAAAATTCTTGCATCTTGCATTTTGGTCATTTGTAAATATTTTTTTAGTCTTAAAAAAGACCCAAATTCTTATTAATTTCAAAATTTTCGGTCTCTAATACCTCCGGTTTTAAAAAAAAACATATCAGTTGAAGGATGAGTTTGGTGAAGGCTATATTGTCCATTGATTTTGGAGATATATGTATTATGGTCATGATTATTACGATTTTTATATAAAAGAATATTAAAAATGGTGGGGTTGGTGAAGAAATGAAGATTTATCGTCAAATATTTCAATTTTTACTTGGACTATTGCTTCGGTTATATCGTCAACATGGGCCCACTCTTCCACCAAAGCCCAATCAATATATCTCTCGCTATCTTCACCAACCCACTCTTCTTCTCTTACCAAACCCATTTCCTTTATTTCCAACCCTACCCCTTTATTTCTCAAGCTTTACACTTTTAGCCCATAACTTTCTTTTTATCCAAATGGATTTGACTGGTCTCCAAAGTTGAATTAAATGGTTGTAGAAATAAAATAAAATTATACGGGTTCAATTGTTCAATTGTTCATATACCGTTGACGTTCAATTGTTCATATACGGGTTCCGTGGTCGTTGGTAATATATATGTCTTTTATGGAACCAAAATAGACCAAATCAACAACAAATGAAGAAATTGTTAGAGTATGATACACTCATATATACCCAAATATAGCATATATTTATAATATAACTTTTGGCTATGTCATTTTACATGATTTTTTTGGCTTATCTATTAAAAGTATCATACAAACTGTTTTTACTTCTTTTTTTTCTTAGAATATATATGCCCAAAATGGAAAAGAACATATGCCAAGGTTGATTTTATCGCTTATATGGTAAAAATTGGAAAAACATACAAATCATTACTTTATTTAATTAAATCATGTGAAGAAACATATTCAATTACGGTAATACGTTATCAAAACATTTTTTTTTACATTAATTGTTACATTTTTTTTTTTTGCAAATATTCTTAAATAACCATTCTTTTTTTATTTACTATAATTAACATAAAAATAAATAAAATATAACATTTCAACAAAGAAATTTGCTTATGAAAAATACAAAATCCAGTTAATTTTTCAGAAAAATACAAATTTGCTTATAAATATATTACCACTAGTTTATGTGATTTTAAAAGAAAGAAATGCAGCTTACCAAACGCAACGTGAAAATTTGAGAAACCCATACTCAAAAAAGATTAAATGACAAAATCACCCTCAGCAAAATCATGAAACAACAACACTAACATTTTCACCAACCCCACCGTCTACTCCGGTGAATTGTCTATATGAACTCCTCCGATACAACTCCTGTTTCCTTCAGGCCAAAGCCTAAAATTCACACAACCAAAAAAACCAACCTTTTTTTTCCACCTAAATCTTTGAATATCACAATATTTACTATTTACASEQ ID NO: 28-AtCco AOMT promoter polynucleotide sequenceACACATTAAAACAAAAACCATTTCCACATAAAAAAAAACGATCCAGTAAATGAAATAGATTCAAGACCGATCGTCGAGCGGTAGAGAAAGTAAACAAAACAAAGACAGAGAATTGAAGAAACTGTGTACCTGCAAAAATACCAATCAGATGGGTCTCCGCCAAAGTAATCTGCTTAGAAGTTTTGTAAGAAAAAACAATTAAAGGCGTTTCATTTATTGAATTTTCCGGTTGTTTGATTCTCAGGATGAGATTGCCTATTTCCTTCAAAAAAGAACTCTTTAATTTACACAGAAAAGCTCTGAAAATTTCCACAGAAAATGAAGAAAGAAAAGAGCGTAAAAGGGGAAAGAGATGAAATGGGTTATTAAAAAAAGAAGCAGTGGATGAGGGAAGAGAGGATTAAGAGGCGTAGAGATTACATGTGATGAATGATACTATCTTTTCTTACAAACACATTTTCGTGTAATTAAAATTTAATTTGGTTCCAAAGATTTTAATCAAAAGAAGTTTGGTAAATTGAAACAGGCAGACATAATTTATTGTAAAGAGTTTTTATTTATTTATTCATGACGTTGCTTGATGGTGCTTTACCAATTTTCTTCTCCTACGTTAGATTTTTTTCACTTTTTTTTTTGGTGTTTGTAATAAATGTGAAAAATGGACCGTTTAAAAACTTAAAGACGTTTGATTACTATATAAAGTAATTGTTTATAATAGAAAGTTAATTGAGACGTGAAATGGTATAATATTATTGTGTAACAGTTGTGTACACGTAGCTCTCATGCAGTTTTAGTGGACCCATATGGCTTGACTTGTATTCTGTTTTTGGGCTATTAAAGTCCAAAACAGAGACCCCTCTCAAGCCCTTCCTATTAATCCATCTAGCTAATAGAAACTATAAACGTGTCCTCTCTCTCAATTAAATAAGCTAGAAACATACTCAACCATTCGCATTACGCACTTCATAGCGGTAGGTTTAGATTTGTCTAAAATACTTAAAAAAATTTTTGTCTAAGTTGTTGTCCGTTACAAAGTTTTTTTCTTTGTGACAACTTGACAACATTGACAAATAGAAAAATAAATTTCGATGAAACCTATGAAATGGGCTATGGCCCAACTAAAAAGAGTGGGAAATTAAAGATGGGATGGTTCAAGTGTATACTTCGAACTTCCGACATTAGGGTCAAAGGATTTTTAAAAGGCAACCATTTGTTCCACTTTCTCGAACAAAAACGAGCCATTTATTAATATATAGTACGGCTGAATTGGTTTTGTTCGTCATTGTGTAAACACAAAGTCATTCGAATTATGTTAGGGTCCGTTGATAATATAGACGGCCCATCCCACGCACATATTAAGTGTTCAACTCCATAGAATATCATATGGGACACTGTTTTTAATTTATAATCACCATTTAAAATGTTTAAATGTTTATGCAAATTGGATGGCTTCTTCACACAACATTTATTTATTGGCCTTTCATTCCATCAAAGTAAAATAGCTTTTCAAATACATTATACTCTATACTCCTATACATGTAAATAACCATATGCATATATATTTTTTTCAAATATAGGTCAACGCCATTTAATATAATTTTAAAAAAATTTGTTCGGAAAATATCACATTTCTTTCACTAGACAAGCCTTGTTACCACACAATGTATCAATATGATCTAAAGGGCAAACGAAAGATCCTGACATGAAACGTTTAATTCTCATTTTCTCCAAATTTTATTTTTTATGTGAAGTAGATAAATTAGTATATATATATATATACCAAACTAGTGTGTTATGTTATGGCAAATGTTATATCAATTCGAAGGTTCCGCTATTGCAATATTCATTAATTTTTTCATACCAATACTATTTTTCTTTCTCTTTTATTTTGTTTTTTAATAAATAAAAGAAATTAAGGATGATTAGTAAGGAAGTCGCCTACCAAGAGATTCACCTACCACGGTACACTTCAACACCGAAGCAGAGTTGTTGAATCCACTTTTTATTCCCTTCTCTAATCTCTACTCACCAAGTCTCCACTTTTTTTTCTCTTTATTATATACATTTAAATTATTTAATATACGCCAACTACATACATATCCAGTGTAATTTCTCGTTACGTCACACCCCTTTCGTAATCGTCTAATTTCAGAAAAATATCCAGAGGTTTAAATACATATTCCCATCATTAAATCTAGACATAAACACATCATACTCACAAAATTTGGCAGCAAACAGTTACTACAGACCCATAAATGAAAAAACGTATTCACTTGTTTTCAATTTTCACATAACCACTTCCCTGAGTTTGGTCTCAATTTGATTGCCCCGCCGAGGCATTACTACGCCAAGTGCGATTAAGGTCCCATACAGTGTAACGGGACCCACTATAAGACAGCGACCGACCAATTGCGTGTTAGGAGAGTTTCACCAACCCCGGACCGGTTTTTACCGGATATAACAGAACCGGTACGAACCGGTCTCATTATCTTCCATCTTCTTTATATAGACCTCATGCCATGTGTGTGACTCACCAAGAAAAACACAATCGTTTAATCTCACCCAAGAAGACAAAAACACAGAGAGAGAAAGAGAGAGAASEQ ID NO: 29-TcPAM amino acid sequence (Taxus chinensis phenylalanineaminomutase; AAT47186)MGFAVESRSHVKDILGLINTFNEVKKITVDGTTPITVAHVAALARRHDVKVALEAEQCRARVETCSSWVQRKAEDGADIYGVTTGFGACSSRRTNQLSELQESLIRCLLAGVFTKGCASSVDELPATATRSAMLLRLNSFTYGCSGIRWEVMEALEKLLNSNVSPKVPLRGSVSASGDLIPLAYIAGLLIGKPSVVARIGDDVEVPAPEALSRVGLRPFKLQAKEGLALVNGTSFATALASTVMYDANVLLLLVETLCGMFCEVIFGREEFAHPLIHKVKPHPGQIESAELLEWLLRSSPFQDLSREYYSIDKLKKPKQDRYALRSSPQWLAPLVQTIRDATTTVETEVNSANDNPIIDHANDRALHGANFQGSAVGFYMDYVRIAVAGLGKLLFAQFTELMIEYYSNGLPGNLSLGPDLSVDYGLKGLDIAMAAYSSELQYLANPVTTHVHSAEQHNQDINSLALISARKTEEALDILKLMIASHLTAMCQAVDLRQLEEALVKVVENVVSTLADECGLPNDTKARLLYVAKAVPVYTYLESPCDPTLPLLLGLEQSCFGSILALHKKDGIETDTLVDRLAEFEKRLSDRLENEMTAVRVLYEKKGHKTADNNDALVRIQGSRFLPFYRFVREELDTGVMSARREQTPQEDVQKVFDAIADGRITVPLLHCLQGFLGQPNGCANGVESFQSVWNKSASEQ ID NO: 30-PDC amino acid sequence (Pediococcus pentosaceus Phenylacrylicdecarboxylase; CAC16794)MEKTFKTLDDFLGTHFIYTYDNGWEYEWYAKNDHTVDYRIHGGMVAGRWVKDQEAHIAMLTEGIYKVAWTEPTGTDVALDFVPNEKKLNGTIFFPKWVEEHPEITVTFQNEHIDLMEESREKYETYPKLVVPEFATITYMGDAGQDNDEVIAEAPYEGMTDDIRAGKYFDENYKRINKSEQ ID NO: 31-CHS amino acid sequence (Physcomitrella patens chalconesynthase;ABB84527)MASAGDVTRAALPRAQPRAEGPACVLGIGTAVPPAEFLQSEYPDFFFNITNCGEKEALKAKFKRICDKSGIRKRHMFLTEEVLKANPGICTYMEPSLNVRHDIVVVQVPKLAAEAAQKAIKEWGGRKSDITHIVFATTSGVNMPGADHALAKLLGLKPTVKRVMMYQTGCFGGASVLRVAKDLAENNKGARVLAVASEVTAVTYRAPSENHLDGLVGSALFGDGAGVYVVGSDPKPEVEKPLFEVHWAGETILPESDGAIDGHLTEAGLIFHLMKDVPGLISKNIEKFLNEARKPVGSPAWNEMFWAVHPGGPAILDQVEAKLKLTKDKMQGSRDILSEFGNMSSASVLFVLDQIRHRSVKMGASTLGEGSEFGFFIGFGPGLTLEVLVLRAAPNSASEQ ID NO: 32-CHS amino acid sequence (Arabidopsis thaliana chalcone synthase;AAA32771)MVMAGASSLDEIRQAQRADGPAGILAIGTANPENHVLQAEYPDYYFRITNSEHMTDLKEKFKRMCDKSTIRKRHMHLTEEFLKENPHMCAYMAPSLDTRQDIVVVEVPKLGKEAAVKAIKEWGQPKSKITHVVFCTTSGVDMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRIAKDLAENNRGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFSDGAAALIVGSDPDTSVGEKPIFEMVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIVKSLDEAFKPLGISDWNSLFWIAHPGGPAILDQVEIKLGLKEEKMRATRHVLSEYGNMSSACVLFILDEMRRKSAKDGVATTGEGLEWGVLFGFGPGLTVETVVLHSVPLSEQ ID NO: 33-SPS amino acid sequence (Vitis vinifera stilbene synthase; ABE68894)MASVEEFRNAQRAKGPATILAIGTATPDHCVYQSDYADFYFRVTKSEHMTALKKKFNRICDKSMIKKRYIHLTEEMLEEHPNIGAYMAPSLNIRQEIITAEVPKLGKEAALKALKEWGQPKSKITHLVFCTTSGVEMPGADYKLANLLGLEPSVRRVMLYHQGCYAGGTVLRTAKDLAENNAGARVLVVCSEITVVTFRGPSEDALDSLVGQALFGDGSAAVIVGSDPDISIERPLFQLVSAAQTFIPNSAGAIAGNLREVGLTFHLWPNVPTLISENIEKCLTQAFDPLGISDWNSLFWIAHPGGPAILDAVEAKLNLDKKKLEATRHVLSEYGNMSSACVLFILDEMRKKSLKGERATTGEGLDWGVLFGFGPGLTIETVVLHSIPMVTNSEQ ID NO: 34-CUS amino acid sequence (Oryza sativa curcuminoid synthase shortversion; 3OIT_A)MRRSQRADGLAAVLAIGTANPPNCVTQEEIPDFYFRVTNSDHLTALKDKFKRICQEMGVQRRYLHHTEEMLSAHPEFVDRDAPSLDARLDIAADAVPELAAEAAKKAIAEWGRPAADITHLVVTTNSGAHVPGVDFRLVPLLGLRPSVRRTMLHLNGCFAGCAALRLAKDLAENSRGARVLVVAAELTLMYFTGPDEGCFRTLLVQGLFGDGAAAVIVGADADDVERPLFEIVSAAQTIIPESDHALNMRFTERRLDGVLGRQVPGLIGDNVERCLLDMFGPLLGGDGGGGWNDLFWAVHPGSSTIMDQVDAALGLEPGKLAASRRVLSDYGNMSGATVIFALDELRRQRKEAAAAGEWPELGVMMAFGPGMTVDAMLLHATSHVNSEQ ID NO: 35-CUS amino acid sequence (Oryza sativa curcuminoid synthase long version;3ALE_A)MAPTTTMGSALYPLGEMRRSQRADGLAAVLAIGTANPPNCVTQEEIPDFYFRVTNSDHLTALKDKFKRICQEMGVQRRYLHHTEEMLSAHPEFVDRDAPSLDARLDIAADAVPELAAEAAKKAIAEWGRPAADITHLVVTTNSGAHVPGVDFRLVPLLGLRPSVRRTMLHLNGCFAGCAALRLAKDLAENSRGARVLVVAAELTLMYFTGPDEGCFRTLLVQGLFGDGAAAVIVGADADDVERPLFEIVSAAQTIIPESDHALNMRFTERRLDGVLGRQVPGLIGDNVERCLLDMFGPLLGGDGGGGWNDLFWAVHPGSSTIMDQVDAALGLEPGKLAASRRVLSDYGNMSGATVIFALDELRRQRKEAAAAGEWPELGVMMAFGPGMTVDAMLLHATSHVNSEQ ID NO: 36-BAS amino acid sequence (Rheum palmatum benzalacetone synthase; AAK82824)MATEEMKKLATVMAIGTANPPNCYYQADFPDFYFRVTNSDHLINLKQKFKRLCENSRIEKRYLHVTEEILKENPNIAAYEATSLNVRHKMQVKGVAELGKEAALKAIKEWGQPKSKITHLIVCCLAGVDMPGADYQLTKLLDLDPSVKRFMFYHLGCYAGGTVLRLAKDIAENNKGARVLIVCSEMTTTCFRGPSETHLDSMIGQAILGDGAAAVIVGADPDLTVERPIFELVSTAQTIVPESHGAIEGHLLESGLSFHLYKTVPTLISNNIKTCLSDAFTPLNISDWNSLFWIAHPGGPAILDQVTAKVGLEKEKLKVTRQVLKDYGNMSSATVFFIMDEMRKKSLENGQATTGEGLEWGVLFGFGPGITVETVVLRSVPVISSEQ ID NO: 37-AtPAP1 amino acid sequence (Arabidopsis thaliana R2R3 Myb transcriptionfactor, AtMyb75; AAG42001)MEGSSKGLRKGAWTTEEDSLLRQCINKYGEGKWHQVPVRAGLNRCRKSCRLRWLNYLKPSIKRGKLSSDEVDLLLRLHRLLGNRWSLIAGRLPGRTANDVKNYWNTHLSKKHEPCCKIKMKKRDITPIPTTPALKNNVYKPRPRSFTVNNDCNHLNAPPKVDVNPPCLGLNINNVCDNSIIYNKDKKKDQLVNNLIDGDNMWLEKFLEESQEVDILVPEATTTEKGDTLAFDVDQLWSLFDGETVKFDSEQ ID NO: 38-AtPAP2 amino acid sequence (Arabidopsis thaliana R2R3 Myb transcriptionfactor, AtMyb90; AAG42002)MEGSSKGLRKGAWTAEEDSLLRLCIDKYGEGKWHQVPLRAGLNRCRKSCRLRWLNYLKPSIKRGRLSNDEVDLLLRLHKLLGNRWSLIAGRLPGRTANDVKNYWNTHLSKKHESSCCKSKMKKKNIISPPTTPVQKIGVFKPRPRSFSVNNGCSHLNGLPEVDLIPSCLGLKKNNVCENSITCNKDDEKDDFVNNLMNGDNMWLENLLGENQEADAIVPEATTAEHGATLAFDVEQLWSLFDGETVELDSEQ ID NO: 39-AtTT2 amino acid sequence (Arabidopsis thaliana R2R3 Myb transcriptionfactor, AtMyb123; AED93980)MGKRATTSVRREELNRGAWTDHEDKILRDYITTHGEGKWSTLPNQAGLKRCGKSCRLRWKNYLRPGIKRGNISSDEEELIIRLHNLLGNRWSLIAGRLPGRTDNEIKNHWNSNLRKRLPKTQTKQPKRIKHSTNNENNVCVIRTKAIRCSKTLLFSDLSLQKKSSTSPLPLKEQEMDQGGSSLMGDLEFDFDRIHSEFHFPDLMDFDGLDCGNVTSLVSSNEILGELVPAQGNLDLNRPFTSCHHRGDDEDWLRDFTCSEQ ID NO: 40-NtAn2 amino acid sequence (Nicotiana tabacum R2R3 Myb transcriptionfactor; ACO52470)MNICTNKSSSGVKKGAWTEEEDVLLKKCIEKYGEGKWHQVPLRAGLNRCRKSCRLRWLNYLRPHIKRGDFSFDEVDLILRLHKLLGNRWSLIAGRLPGRTANDVKNYWNSHLRKKLIAPHDQKESKQKAKKITIFRPRPRTFSKTNTCVKSNTNTVDKDIEGSSEIIRFNDNLKPTTEELTDDGIQWWADLLANNYNNNGIEEADNSSPTLLHEEMPLLSSEQ ID NO: 41-MtLAP1 amino acid sequence (Medicago truncatula R2R3 Myb transcriptionfactor; ACN79541)MENTGGVRKGAWTYKEDELLKACINTYGEGKWNLVPQRSGLNRCRKSCRLRWLNYLSPNINRGRFSEDEEDLILRLHKLLGNRWSLIAGRLPGRTANDVKNYWHTNLAKKVVSEKEEEKENDKPKETMKAHEVIKPRPITLSSHSNWLKGKNSIPRDLDYSENMASNQIGRECASTSKPDLGNAPIPCEMWCDSLWNLGEHVDSEKIGSCSSLQEENLMEFPNVDDDSFWDFNLCDLNSLWDLPSEQ ID NO: 42-ZmMYB-C amino acid sequence (Zea mays R2R3 Myb transcriptionfactor; AAK09326)MGRRACCAKEGVKRGAWTSKEDDALAAYVKAHGEGKWREVPQKAGLRRCGKSCRLRWLNYLRPNIRRGNISYDEEDLIIRLHRLLGNRWSLIAGRLPGRTDNEIKNYWNSTLGRRAGAGAGAGGSWVVVAPDTGSHATPATSGACETGQNSAAHRADPDSAGTTTTSAAAVWAPKAVRCTGGLFFFHRDTTPAHAGETATPMAGGGGGGGGEAGSSDDCSSASVSLRVGSHDEPCFSGDGDGDWMDDVRALASFLESDEDWLRCQTAGQLASEQ ID NO: 43-ZmMYC-Lc amino acid sequence (Zea mays BHLH transcription factor;ABD72707)MALSASRVQQAEELLQRPAERQLMRSQLAAAARSINWSYALFWSISDTQPGVLTWTDGFYNGEVKTRKISNSVELTSDQLVMQRSDQLRELYEALLSGEGDRRAAPARPAGSLSPEDLGDTEWYYVVSMTYAFRPGQGLPGRSFASDEHVWLCNAHLAGSKAFPRALLAKSASIQSILCIPVMGGVLELGTTDTVPEAPDLVSRATAAFWEPQCPSSSPSGRANETGEAAADDGTFAFEELDHNNGMDDIEAMTAAGGHGQEEELRLREAEALSDDASLEHITKEIEEFYSLCDEMDLQALPLPLEDGWTVDASNFEVPCSSPQPAPPPVDRATANVAADASRAPVYGSRATSFMAWTRSSQQSSCSDDAAPAAVVPAIEEPQRLLKKVVAGGGAWESCGGATGAAQEMSGTGTKNHVMSERKRREKLNEMFLVLKSLLPSIHRVNKASILAETIAYLKELQRRVQELESSREPASRPSETTTRLITRPSRGNNESVRKEVCAGSKRKSPELGRDDVERPPVLTMDAGTSNVTVTVSDKDVLLEVQCRWEELLMTRVFDAIKSLHLDVLSVQASAPDGFMGLKIRAQFAGSGAVVPWMISEALRKAIGKRSEQ ID NO: 44-AtTT8 amino acid sequence (Arabidopsis thaliana BHLH transcriptionfactor; AEE82802)MDESSIIPAEKVAGAEKKELQGLLKTAVQSVDWTYSVFWQFCPQQRVLVWGNGYYNGAIKTRKTTQPAEVTAEEAALERSQQLRELYETLLAGESTSEARACTALSPEDLTETEWFYLMCVSFSFPPPSGMPGKAYARRKHVWLSGANEVDSKTFSRAILAKSAKIQTVVCIPMLDGVVELGTTKKVREDVEFVELTKSFFYDHCKTNPKPALSEHSTYEVHEEAEDEEEVEEEMTMSEEMRLGSPDDEDVSNQNLHSDLHIESTHTLDTHMDMMNLMEEGGNYSQTVTTLLMSHPTSLLSDSVSTSSYIQSSFATWRVENGKEHQQVKTAPSSQWVLKQMIFRVPFLHDNTKDKRLPREDLSHVVAERRRREKLNEKFITLRSMVPFVTKMDKVSILGDTIAYVNHLRKRVHELENTHHEQQHKRTRTCKRKTSEEVEVSIIENDVLLEMRCEYRDGLLLDILQVLHELGIETTAVHTSVNDHDFEAEIRAKVRGKKASIAEVKRAIHQVIIHDTNLSEQ ID NO: 45-VyMyc1 amino acid sequence (Vitis vinifera BHLH transcription factor;ACC68685)MAAPPNSRLQSMLQSAVQSVRWTYSLFWQICPQQGILVWGDGYYNGAIKTRKTVQPMEVSAEEASLQRSQQLRELYESLSAGETNQPARRPCAALSPEDLTESEWFYLMCVSFSFPPGVGLPGKAYAKRHHIWLAGANEVDSKVFSRAILAKSARVQTVVCIPLMDGVVEFGTTEKVQEDLGFVQHVKSFFTDHHLHNHPPKPALSEHSTSNPATSSDHSRFHSPPIQAAYAAADPPASNNQEEEEEEEEEEEEEEEEEEEEEEEEAESDSEAETGRNNRRVRTQNTGTEGVAGSHTAAEPSELIQLEMSEGIRLGSPDDGSNNLDSDFHMLAVSQPGSSVDHQRRADSYRAESARRWPMLQDPLCSSGLQQPPPQPPTGPPPLDELSQEDTHYSQTVSTILQHQPNRWSESSSSGCIAPYSSQSAFAKWTTRCDHHHHPMAVEGTSQWLLKYILFSVPFLHTKYRDENSPKSRDGDSAGRFRKGTPQDELSANHVLAERRRREKLNERFIILRSLVPFVTKMDKASILGDTIEYVKQLRKKIQDLEARTRQMEVEQRSRGSDSVRSKEHRIGSGSVDRNRAVVAGSDKRKLRIVEGSTGAKPKVVDSPPAAVEGGTTTVEVSIIESDALLEMQCPYREGLLLDVMQMLRELRLETTIVQSSLINGVFVAELRAKVKENASGKKASIMEVKRAINQIIPQC

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1.-16. (canceled)
 17. A method of engineering a plant having reducedlignin content, the method comprising: introducing into the plant anexpression cassette comprising a polynucleotide that encodes a proteinthat reduces the amount of cytosolic and/or plastidial phenylalaninethat is available for a lignin biosynthesis pathway in the plant,wherein the polynucleotide is operably linked to a heterologouspromoter, and wherein the promoter is a secondary cell wall-specificpromoter, a fiber cell-specific promoter, or a promoter from a gene inthe lignin biosynthesis pathway; and culturing the plant underconditions in which the protein is expressed.
 18. The method of claim17, wherein the protein is phenylacetaldehyde synthase (PAAS) orphenylalanine aminomutase (PAM).
 19. The method of claim 18, wherein theprotein is PAAS and has at least 95% amino acid sequence identity to SEQID NO:10.
 20. A plant engineered by the method of claim
 17. 21. A plantcell from the plant of claim 20, wherein plant cell comprises theexpression cassette.
 22. A seed, flower, leaf, or fruit from the plantof claim 20, wherein the seed, flower, leaf, or fruit comprises theexpression cassette.
 23. A method of engineering a plant having reducedlignin content, the method comprising: introducing into the plant anexpression cassette comprising a polynucleotide that encodes a proteinthat reduces the amount of coumaroyl-CoA, caffeoyl-CoA, and/orferuloyl-CoA that is available for a lignin biosynthesis pathway in theplant, wherein the polynucleotide is operably linked to a heterologouspromoter, and wherein the promoter is a secondary cell wall-specificpromoter, a fiber cell-specific promoter, or a promoter from a gene inthe lignin biosynthesis pathway; and culturing the plant underconditions in which the protein is expressed.
 24. The method of claim23, wherein the protein is 2-oxoglutarate-dependent dioxygenase (C2′H)or curcuminoid synthase (CUS).
 25. The method of claim 24, wherein theprotein is 2-oxoglutarate-dependent dioxygenase (C2′H) and has at least95% amino acid sequence identity to SEQ ID NO:14; or the protein iscurcuminoid synthase and has at least 95% amino acid sequence identityto SEQ ID NO:34 or SEQ ID NO:35.
 26. A plant engineered by the method ofclaim
 23. 27. A plant cell from the plant of claim 26, wherein plantcell comprises the expression cassette.
 28. A seed, flower, leaf, orfruit from the plant of claim 26, wherein the seed, flower, leaf, orfruit comprises the expression cassette.